Stimulation of cell-surface urokinase-type plasminogen activator activity and cell migration in vascular endothelial cells by a novel hexapeptide analogue of neurotensin.

PubMed

Ushiro, S; Mizoguchi, K; Yoshida, S; Jimi, S; Fujiwara, T; Yoshida, M; Wei, E T; Kitabgi, P; Amagaya, S; Ono, M; Kuwano, M

1997-12-01

To investigate if neurotensin (NT) could induce activation of urokinase-type plasminogen activator (uPA) in vascular endothelial cells, we utilized the acetyl-NT (8-13) analogue, TJN-950, in which the C-terminal leucine is reduced to leucinol. TJN-950 inhibited the binding of 125I-NT to membranes of newborn rat brains and of COS-7 cells transfected with rat NT receptor cDNA, but at 10(4) higher doses than NT (8-13). However, TJN-950 was as effective as NT in inducing the fibrinolytic activity in bovine vascular aortic and human umbilical vein endothelial cells, and enhanced the migration of vascular endothelial cells. Moreover, administration of TJN-950 induced neovascularization in the rat cornea in vivo. TJN-950 had no effect on expression of uPA, plasminogen activator inhibitor-1 or uPA receptor mRNA. The binding of 125I-TJN-950 to cell membranes was blocked by unlabeled uPA and TJN-950, but not the amino-terminal or 12-32 fragment of uPA. TJN-950 may enhance uPA activity in vascular endothelial cells by interacting with the uPA receptor, resulting in induction of angiogenesis.

Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor.

PubMed Central

Colman, R W; Pixley, R A; Najamunnisa, S; Yan, W; Wang, J; Mazar, A; McCrae, K R

1997-01-01

The urokinase receptor (uPAR) binds urokinase-type plasminogen activator (u-PA) through specific interactions with uPAR domain 1, and vitronectin through interactions with a site within uPAR domains 2 and 3. These interactions promote the expression of cell surface plasminogen activator activity and cellular adhesion to vitronectin, respectively. High molecular weight kininogen (HK) also stimulates the expression of cell surface plasminogen activator activity through its ability to serve as an acquired receptor for prekallikrein, which, after its activation, may directly activate prourokinase. Here, we report that binding of the cleaved form of HK (HKa) to human umbilical vein endothelial cells (HUVEC) is mediated through zinc-dependent interactions with uPAR. These occur through a site within uPAR domains 2 and 3, since the binding of 125I-HKa to HUVEC is inhibited by vitronectin, anti-uPAR domain 2 and 3 antibodies and soluble, recombinant uPAR (suPAR), but not by antibody 7E3, which recognizes the beta chain of the endothelial cell vitronectin receptor (integrin alphavbeta3), or fibrinogen, another alphavbeta3 ligand. We also demonstrate the formation of a zinc-dependent complex between suPAR and HKa. Interactions of HKa with endothelial cell uPAR may underlie its ability to promote kallikrein-dependent cell surface plasmin generation, and also explain, in part, its antiadhesive properties. PMID:9294114

Role of the urokinase plasminogen activator receptor in mediating impaired efferocytosis of anti-SSA/Ro-bound apoptotic cardiocytes: Implications in the pathogenesis of congenital heart block.

PubMed

Briassouli, Paraskevi; Komissarova, Elena V; Clancy, Robert M; Buyon, Jill P

2010-08-06

Binding of maternal anti-Ro/La antibodies to cognate antigen expressed on apoptotic cardiocytes decreases clearance by healthy cardiocytes, which may contribute to the development of autoimmune associated congenital heart block and fatal cardiomyopathy. Given recent evidence implicating the urokinase plasminogen activator receptor (uPAR) as a "don't eat me" signal during efferocytosis, experiments addressed whether surface bound anti-Ro antibodies inhibit apoptotic cell removal via an effect on the expression/function of the urokinase-type plasminogen activator protease uPA/uPAR system. As assessed by flow cytometry and confocal microscopy, uPAR colocalizes and interacts with Ro60 on the surface of apoptotic human fetal cardiocytes. Blocking of uPAR enhances phagocytosis of apoptotic cardiocytes by healthy cardiocytes and reverses the anti-Ro60-dependent impaired clearance of apoptotic cardiocytes. Binding of anti-Ro60 antibodies to apoptotic cardiocytes results in increased uPAR expression, as well as enhanced uPA activity. The binding of anti-Ro60 did not alter other surface molecules involved in cell recognition (calreticulin, CD31, or CD47). These data suggest that increased uPAR expression and uPA activity induced by anti-Ro60 binding to the apoptotic fetal cardiocyte provide a molecular basis by which these antibodies inhibit efferocytosis and ultimately lead to scar of the fetal conduction system and working myocardium.

Persons with Quebec platelet disorder have a tandem duplication of PLAU, the urokinase plasminogen activator gene.

PubMed

Paterson, Andrew D; Rommens, Johanna M; Bharaj, Bhupinder; Blavignac, Jessica; Wong, Isidro; Diamandis, Maria; Waye, John S; Rivard, Georges E; Hayward, Catherine P M

2010-02-11

Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder linked to a region on chromosome 10 that includes PLAU, the urokinase plasminogen activator gene. QPD increases urokinase plasminogen activator mRNA levels, particularly during megakaryocyte differentiation, without altering expression of flanking genes. Because PLAU sequence changes were excluded as the cause of this bleeding disorder, we investigated whether the QPD mutation involved PLAU copy number variation. All 38 subjects with QPD had a direct tandem duplication of a 78-kb genomic segment that includes PLAU. This mutation was specific to QPD as it was not present in any unaffected family members (n = 114), unrelated French Canadians (n = 221), or other persons tested (n = 90). This new information on the genetic mutation will facilitate diagnostic testing for QPD and studies of its pathogenesis and prevalence. QPD is the first bleeding disorder to be associated with a gene duplication event and a PLAU mutation.

Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase.

PubMed

López-Alemany, Roser; Longstaff, Colin; Hawley, Stephen; Mirshahi, Massoud; Fábregas, Pere; Jardí, Merce; Merton, Elizabeth; Miles, Lindsey A; Félez, Jordi

2003-04-01

Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells. Copyright 2003 Wiley-Liss, Inc.

Platelets from patients with the Quebec platelet disorder contain and secrete abnormal amounts of urokinase-type plasminogen activator.

PubMed

Kahr, W H; Zheng, S; Sheth, P M; Pai, M; Cowie, A; Bouchard, M; Podor, T J; Rivard, G E; Hayward, C P

2001-07-15

The Quebec platelet disorder (QPD) is an autosomal dominant platelet disorder associated with delayed bleeding and alpha-granule protein degradation. The degradation of alpha-granule, but not plasma, fibrinogen in patients with the QPD led to the investigation of their platelets for a protease defect. Unlike normal platelets, QPD platelets contained large amounts of fibrinolytic serine proteases that had properties of plasminogen activators. Western blot analysis, zymography, and immunodepletion experiments indicated this was because QPD platelets contained large amounts of urokinase-type plasminogen activator (u-PA) within a secretory compartment. u-PA antigen was not increased in all QPD plasmas, whereas it was increased more than 100-fold in QPD platelets (P <.00009), which contained increased u-PA messenger RNA. Although QPD platelets contained 2-fold more plasminogen activator inhibitor 1 (PAI-1) (P <.0008) and 100-fold greater u-PA-PAI-1 complexes (P <.0002) than normal platelets, they contained excess u-PA activity, predominantly in the form of two chain (tcu-PA), which required additional PAI-1 for full inhibition. There was associated proteolysis of plasminogen in QPD platelets, to forms that comigrated with plasmin. When similar amounts of tcu-PA were incubated with normal platelet secretory proteins, many alpha-granule proteins were proteolyzed to forms that resembled degraded QPD platelet proteins. These data implicate u-PA in the pathogenesis of alpha-granule protein degradation in the QPD. Although patients with the QPD have normal to increased u-PA levels in their plasma, without evidence of systemic fibrinogenolysis, their increased platelet u-PA could contribute to bleeding by accelerating fibrinolysis within the hemostatic plug. QPD is the only inherited bleeding disorder in humans known to be associated with increased u-PA.

PEGylated DX-1000: pharmacokinetics and antineoplastic activity of a specific plasmin inhibitor.

PubMed

Devy, Laetitia; Rabbani, Shafaat A; Stochl, Mark; Ruskowski, Mary; Mackie, Ian; Naa, Laurent; Toews, Mark; van Gool, Reinoud; Chen, Jie; Ley, Art; Ladner, Robert C; Dransfield, Daniel T; Henderikx, Paula

2007-11-01

Novel inhibitors of the urokinase-mediated plasminogen (plg) activation system are potentially of great clinical benefit as anticancer treatments. Using phage display, we identified DX-1000 a tissue factor pathway inhibitor-derived Kunitz domain protein which is a specific high-affinity inhibitor of plasmin (pln) (K(i) = 99 pM). When tested in vitro, DX-1000 blocks plasmin-mediated pro-matrix metalloproteinase-9 (proMMP-9) activation on cells and dose-dependently inhibits tube formation, while not significantly affecting hemostasis and coagulation. However, this low-molecular weight protein inhibitor ( approximately 7 kDa) exhibits rapid plasma clearance in mice and rabbits, limiting its potential clinical use in chronic diseases. After site-specific PEGylation, DX-1000 retains its activity and exhibits a decreased plasma clearance. This PEGylated derivative is effective in vitro, as well as potent in inhibiting tumor growth of green fluorescent protein (GFP)-labeled MDA-MB-231 cells. 4PEG-DX-1000 treatment causes a significant reduction of urokinase-type plasminogen activator (uPA) and plasminogen expressions, a reduction of tumor proliferation, and vascularization. 4PEG-DX-1000 treatment significantly decreases the level of active mitogen-activated protein kinase (MAPK) in the primary tumors and reduces metastasis incidence. Together, our results demonstrate the potential value of plasmin inhibitors as therapeutic agents for blocking breast cancer growth and metastasis.

Quality aspects of fibrinolytic agents based on biochemical characterization.

PubMed

Werner, R G; Bassarab, S; Hoffmann, H; Schlüter, M

1991-11-01

The purity, composition and in vitro fibrinolytic activity of four commercially available fibrinolytic agents, alteplase (recombinant tissue plasminogen activator, rt-PA, Actilyse; CAS 105857-23-6), streptokinase, urokinase and anistreplase (ansioyl-plasminogen-streptokinase activator-complex, APSAC), have been compared in this investigation. The fibrinolytic activity was measured in an in vitro thrombolytic assay. In this assay a human blood thrombus is dissolved in an environment of human plasma. This assay is representative for the in vivo situation, where plasminogen activation is also a limiting step in thrombolysis. In the in vitro thrombolytic assay alteplase is about 10 times more effective in clot lysis than either streptokinase or urokinase and more than 300 times more active than anistreplase. In addition, the ratio of active ingredient to total protein content in the preparations was analysed by RP-HPLC, SDS-PAGE, GPC-HPLC and amino acid analysis. The portion of active ingredient per total protein was 99.9% for alteplase, 55% for anistreplase, 20% for urokinase and 1% for streptokinase. This demonstrates that alteplase is the only fibrinolytic agent tested which is essentially free of protein additives of human origine and potential contaminants associated therewith. The superior purity of alteplase compared to the other fibrinolytics was confirmed by SDS-PAGE, RP-HPLC, and HPLC-GPC. Significant levels of aggregates were detected in streptokinase and urokinase preparations, whereas alteplase and anistreplase were essentially free of aggregates. These data demonstrate that there are significant differences in composition, purity and in vitro activity between different fibrinolytic agents.

Transgenic chickens expressing human urokinase-type plasminogen activator.

PubMed

Lee, Sung Ho; Gupta, Mukesh Kumar; Ho, Young Tae; Kim, Teoan; Lee, Hoon Taek

2013-09-01

Urokinase-type plasminogen activator is a serine protease that is clinically used in humans for the treatment of thrombolytic disorders and vascular diseases such as acute ischemic stroke and acute peripheral arterial occlusion. This study explored the feasibility of using chickens as a bioreactor for producing human urokinase-type plasminogen activator (huPA). Recombinant huPA gene, under the control of a ubiquitous Rous sarcoma virus promoter, was injected into the subgerminal cavity of freshly laid chicken eggs at stage X using the replication-defective Moloney murine leukemia virus (MoMLV)-based retrovirus vectors encapsidated with VSV-G (vesicular stomatitis virus G) glycoprotein. A total of 38 chicks, out of 573 virus-injected eggs, hatched and contained the huPA gene in their various body parts. The mRNA transcript of the huPA gene was present in various organs, including blood and egg, and was germ-line transmitted to the next generation. The level of active huPA protein was 16-fold higher in the blood of the transgenic chicken than in the nontransgenic chicken (P < 0.05). The expression of huPA protein in eggs increased from 7.82 IU/egg in the G0 generation to 17.02 IU/egg in the G1 generation. However, huPA-expressing embryos had reduced survival and hatchability at d 18 and 21 of incubation, respectively, and the blood clotting time was significantly higher in transgenic chickens than their nontransgenic counterparts (P < 0.05). Furthermore, adult transgenic rooster showed reduced (P < 0.05) fertility, as revealed by reduced volume of semen ejaculate, sperm concentration, and sperm viability. Taken together, our data suggest that huPA transgenic chickens could be successfully produced by the retroviral vector system. Transgenic chickens, expressing the huPA under the control of a ubiquitous promoter, may not only be used as a bioreactor for pharming of the huPA drug but also be useful for studying huPA-induced bleeding and other disorders.

Interaction of fucoidan with proteases and inhibitors of coagulation and fibrinolysis.

PubMed

Minix, R; Doctor, V M

1997-09-01

The interactions of fucoidan with glutamic plasminogen (Glu-Plg), two-chain tissue plasminogen activator (t-PA), LMwt-urokinase, thrombin, and antithrombin III (AT-III) were investigated using fucoidan-sepharose affinity chromatography. The results showed 1) a high degree of affinity between fucoidan-sepharose and Glu-Plg; Lmwt-urokinase and thrombin while t-Pa and AT-III did not bind with fucoidan-sepharose. 2) The double reciprocal plot for the LMwt-urokinase activation of Glu-Plg showed that plasminogen activator inhibitor (PAI-1) inhibited this reaction in a noncompetitive manner and that the presence of fucoidan decreased Km for this interaction by 50% and increased Kcat by 30-fold, 3) The double reciprocal plot for the t-PA activation of Glu-Plg showed that PAI-1 inhibited this reaction in a competitive manner and that fucoidan in conjunction with 6-aminohexanoic acid (6-AH) increased Kcat for this interaction by 5-fold without affecting Km. 4) Fucoidan enhanced the interaction of thrombin with both AT-III and heparin cofactor II (HC-II) and it was more effective than unfractionated heparin of LMwt-heparin in enhancing the interaction of HC-II with thrombin.

Functional Stability of Plasminogen Activator Inhibitor-1

PubMed Central

Kuru, Pinar; Toksoy Oner, Ebru; Agirbasli, Mehmet

2014-01-01

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of plasminogen activators, such as tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), and a major regulator of the fibrinolytic system. PAI-1 plays a pivotal role in acute thrombotic events such as deep vein thrombosis (DVT) and myocardial infarction (MI). The biological effects of PAI-1 extend far beyond thrombosis including its critical role in fibrotic disorders, atherosclerosis, renal and pulmonary fibrosis, type-2 diabetes, and cancer. The conversion of PAI-1 from the active to the latent conformation appears to be unique among serpins in that it occurs spontaneously at a relatively rapid rate. Latency transition is believed to represent a regulatory mechanism, reducing the risk of thrombosis from a prolonged antifibrinolytic action of PAI-1. Thus, relying solely on plasma concentrations of PAI-1 without assessing its function may be misleading in interpreting the role of PAI-1 in many complex diseases. Environmental conditions, interaction with other proteins, mutations, and glycosylation are the main factors that have a significant impact on the stability of the PAI-1 structure. This review provides an overview on the current knowledge on PAI-1 especially importance of PAI-1 level and stability and highlights the potential use of PAI-1 inhibitors for treating cardiovascular disease. PMID:25386620

Gene expression of fibrinolytic factors urokinase plasminogen activator and plasminogen activator inhibitor-1 in rabbit temporo-mandibular joint cartilage with disc displacement.

PubMed

Zhan, Jing; Gu, Zhi-yuan; Wu, Li-qun; Zhang, Yin-kai; Hu, Ji-an

2005-06-20

The urokinase plasminogen activator system is believed to play an important role in degradation of the extracellular matrix associated with cartilage and bone destruction; however its precise roles in temporomandibular disorders have not yet been clarified. The aims of this study were to investigate the gene expression of fibrinolytic factors urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in the articular cartilage of rabbit temporomandibular joint (TMJ) with disc displacement (DD) and to probe the relationship between fibrinolytic activity and cartilage remodeling. Disc displacement of right joints was performed in 36 of 78 rabbits under investigation. The animals were sacrificed at 4 days and 1, 2, 4, 8 and 12 weeks after surgery, respectively. The right joints of these animals were harvested and processed for the examination of mRNA expression of uPA and PAI-1 in articular cartilage using in situ hybridization techniques. The expression of uPA and PAI-1 was co-expressed weakly in the chondrocytes from transitive zone to hypertrophic zone and mineralized zone, while no hybridizing signals were shown in proliferative zone and superficial zone in control rabbits. The most striking was the up-regulation of uPA and PAI-1 mRNA in 4-day rabbits postoperatively at the onset of cartilage degeneration. The strongest hybridizing signals for uPA and PAI-1 were seen in 2-week rabbits postoperatively. After 2 weeks, the expression of uPA and PAI-1 began to decrease and reached nearly normal level at 12 weeks. The expression of the uPA/PAI-1 system coincides with the pathological changes in condylar cartilage after DD. The uPA/PAI-1 system may be one of the essential mediators in articular cartilage remodeling.

Highly potent fibrinolytic serine protease from Streptomyces.

PubMed

Uesugi, Yoshiko; Usuki, Hirokazu; Iwabuchi, Masaki; Hatanaka, Tadashi

2011-01-05

We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis. Copyright © 2010 Elsevier Inc. All rights reserved.

Urokinase-type plasminogen activator receptor (uPAR) ligation induces a raft-localized integrin signaling switch that mediates the hypermotile phenotype of fibrotic fibroblasts.

PubMed

Grove, Lisa M; Southern, Brian D; Jin, Tong H; White, Kimberly E; Paruchuri, Sailaja; Harel, Efrat; Wei, Ying; Rahaman, Shaik O; Gladson, Candece L; Ding, Qiang; Craik, Charles S; Chapman, Harold A; Olman, Mitchell A

2014-05-02

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

Characterization of the Annonaceous acetogenin, annonacinone, a natural product inhibitor of plasminogen activator inhibitor-1

NASA Astrophysics Data System (ADS)

Pautus, Stéphane; Alami, Mouad; Adam, Fréderic; Bernadat, Guillaume; Lawrence, Daniel A.; de Carvalho, Allan; Ferry, Gilles; Rupin, Alain; Hamze, Abdallah; Champy, Pierre; Bonneau, Natacha; Gloanec, Philippe; Peglion, Jean-Louis; Brion, Jean-Daniel; Bianchini, Elsa P.; Borgel, Delphine

2016-11-01

Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of the tissue type and urokinase type plasminogen activators. High levels of PAI-1 are correlated with an increased risk of thrombotic events and several other pathologies. Despite several compounds with in vitro activity being developed, none of them are currently in clinical use. In this study, we evaluated a novel PAI-1 inhibitor, annonacinone, a natural product from the Annonaceous acetogenins group. Annonacinone was identified in a chromogenic screening assay and was more potent than tiplaxtinin. Annonacinone showed high potency ex vivo on thromboelastography and was able to potentiate the thrombolytic effect of tPA in vivo in a murine model. SDS-PAGE showed that annonacinone inhibited formation of PAI-1/tPA complex via enhancement of the substrate pathway. Mutagenesis and molecular dynamics allowed us to identify annonacinone binding site close to helix D and E and β-sheets 2A.

Regulation of intrapleural fibrinolysis by urokinase-α-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

PubMed Central

Mazar, Andrew P.; Koenig, Kathy; Kurdowska, Anna K.; Idell, Steven

2009-01-01

The proenzyme single-chain urokinase plasminogen activator (scuPA) more effectively resolved intrapleural loculations in rabbits with tetracycline (TCN)-induced loculation than a range of clinical doses of two-chain uPA (Abbokinase) and demonstrated a trend toward greater efficacy than single-chain tPA (Activase) (Idell S et al., Exp Lung Res 33: 419, 2007.). scuPA more slowly generates durable intrapleural fibrinolytic activity than Abbokinase or Activase, but the interactions of these agents with inhibitors in pleural fluids (PFs) have been poorly understood. PFs from rabbits with TCN-induced pleural injury treated with intrapleural scuPA, its inactive Ser195Ala mutant, Abbokinase, Activase, or vehicle, were analyzed to define the mechanism by which scuPA induces durable fibrinolysis. uPA activity was elevated in PFs of animals treated with scuPA, correlated with the ability to clear pleural loculations, and resisted (70–80%) inhibition by PAI-1. α-macroglobulin (αM) but not urokinase receptor complexes immunoprecipitated from PFs of scuPA-treated rabbits retained uPA activity that resists PAI-1 and activates plasminogen. Conversely, little plasminogen activating or enzymatic activity resistant to PAI-1 was detectable in PFs of rabbits treated with Abbokinase or Activase. Consistent with these findings, PAI-1 interacts with scuPA much slower than with Activase or Abbokinase in vitro. An equilibrium between active and inactive scuPA (kon = 4.3 h−1) limits the rate of its inactivation by PAI-1, favoring formation of complexes with αM. These observations define a newly recognized mechanism that promotes durable intrapleural fibrinolysis via formation of αM/uPA complexes. These complexes promote uPA-mediated plasminogen activation in scuPA-treated rabbits with TCN-induced pleural injury. PMID:19666776

Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

PubMed

Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

2001-07-01

The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

Inhibitory effect of berberine on the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, P.-L.; Hsieh, Y.-S.; Wang, C.-J.

2006-07-01

Berberine, a compound isolated from medicinal herbs, has been reported with many pharmacological effects related to anti-cancer and anti-inflammation capabilities. In this study, we observed that berberine exerted a dose- and time-dependent inhibitory effect on the motility and invasion ability of a highly metastatic A549 cells under non-cytotoxic concentrations. In cancer cell migration and invasion process, matrix-degrading proteinases are required. A549 cell treated with berberine at various concentrations showed reduced ECM proteinases including matrix metalloproteinase-2 (MMP2) and urokinase-plasminogen activator (u-PA) by gelatin and casein zymography analysis. The inhibitory effect is likely to be at the transcriptional level, since the reductionmore » in the transcripts levels was corresponding to the proteins. Moreover, berberine also exerted its action via regulating tissue inhibitor of metalloproteinase-2 (TIMP-2) and urokinase-plasminogen activator inhibitor (PAI). The upstream mediators of the effect involved c-jun, c-fos and NF-{kappa}B, as evidenced by reduced phosphorylation of the proteins. These findings suggest that berberine possesses an anti-metastatic effect in non-small lung cancer cell and may, therefore, be helpful in clinical treatment.« less

Hepatitis virus infection affects DNA methylation in mice with humanized livers.

PubMed

Okamoto, Yasuyuki; Shinjo, Keiko; Shimizu, Yasuhiro; Sano, Tsuyoshi; Yamao, Kenji; Gao, Wentao; Fujii, Makiko; Osada, Hirotaka; Sekido, Yoshitaka; Murakami, Shuko; Tanaka, Yasuhito; Joh, Takashi; Sato, Shinya; Takahashi, Satoru; Wakita, Takaji; Zhu, Jingde; Issa, Jean-Pierre J; Kondo, Yutaka

2014-02-01

Cells of tumors associated with chronic inflammation frequently have altered patterns of DNA methylation, including hepatocellular carcinomas. Chronic hepatitis has also been associated with aberrant DNA methylation, but little is known about their relationship. Pyrosequencing was used to determine the methylation status of cultured Huh7.5.1 hepatoma cells after hepatitis C virus (HCV) infection. We also studied mice with severe combined immunodeficiency carrying the urokinase-type plasminogen activator transgene controlled by an albumin promoter (urokinase-type plasminogen activator/severe combined immunodeficient mice), in which up to 85% of hepatocytes were replaced by human hepatocytes (chimeric mice). Mice were given intravenous injections of hepatitis B virus (HBV) or HCV, liver tissues were collected, and DNA methylation profiles were determined at different time points after infection. We also compared methylation patterns between paired samples of hepatocellular carcinomas and adjacent nontumor liver tissues from patients. No reproducible changes in DNA methylation were observed after infection of Huh7.5.1 cells with HCV. Livers from HBV- and HCV-infected mice had genome-wide, time-dependent changes in DNA methylation, compared with uninfected urokinase-type plasminogen activator/severe combined immunodeficient mice. There were changes in 160 ± 63 genes in HBV-infected and 237 ± 110 genes in HCV-infected mice. Methylation of 149 common genes increased in HBV- and HCV-infected mice; methylation of some of these genes also increased in hepatocellular carcinoma samples from patients compared with nontumor tissues. Expression of Ifng, which is expressed by natural killer cells, increased significantly in chimeric livers, in concordance with induction of DNA methylation, after infection with HBV or HCV. Induction of Ifng was reduced after administration of an inhibitor of natural killer cell function (anti-asialo GM1). In chimeric mice with humanized livers, infection with HBV and HCV appears to activate a natural kill cell-dependent innate immune response. This contributes to the induction and accumulation of aberrant DNA methylation in human hepatocytes. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

PubMed

De Lorenzi, Valentina; Sarra Ferraris, Gian Maria; Madsen, Jeppe B; Lupia, Michela; Andreasen, Peter A; Sidenius, Nicolai

2016-07-01

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process. © 2016 The Authors.

Inhibition of PAI-1 Antiproteolytic Activity Against tPA by RNA Aptamers

PubMed Central

Damare, Jared; Brandal, Stephanie

2014-01-01

Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) inhibits the plasminogen activators: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). Elevated levels of PAI-1 have been correlated with an increased risk for cardiovascular disease. Pharmacologically suppressing PAI-1 might prevent, or successfully treat PAI-1 related vascular diseases. This can potentially be accomplished by using small RNA molecules (aptamers). This study's goal is to develop RNA aptamers to a region of PAI-1 that will prevent the ability of PAI-1 to interact with the plasminogen activators. The aptamers were generated through a systematic evolution of ligands via exponential enrichment approach that ensures the creation of RNA molecules that bind to our target protein, PAI-1. In vitro assays were used to determine the effect of these aptamers on PAI-1's inhibitory activity. Three aptamers that bind to PAI-1 with affinities in the nanomolar range were isolated. The aptamer clones R10-4 and R10-2 inhibited PAI-1's antiproteolytic activity against tPA and disrupted PAI-1's ability to form a stable covalent complex with tPA. Increasing aptamer concentrations correlated positively with an increase in cleaved PAI-1. To the best of our knowledge, this is the first report of RNA molecules that inhibit the antiproteolytic activity of PAI-1. PMID:24922319

Intracellular activation of the fibrinolytic cascade in the Quebec Platelet Disorder.

PubMed

Sheth, Prameet M; Kahr, Walter H A; Haq, M Anwar; Veljkovic, Dragoslava Kika; Rivard, Georges E; Hayward, Catherine P M

2003-08-01

The Quebec Platelet Disorder (QPD) is an unusual bleeding disorder associated with increased platelet stores of urokinase-type plasminogen activator (u-PA) and proteolysis of platelet alpha-granule proteins. The increased u-PA and proteolyzed plasminogen in QPD platelets led us to investigate possible contributions of intracellular plasmin generation to QPD alpha-granule proteolysis. ELISA indicated there were normal amounts of plasminogen and plasmin-alpha(2)-antiplasmin (PAP) complexes in QPD plasmas. Like normal platelets, QPD platelets contained only a small proportion of the blood plasminogen, however, they contained an increased amount of PAP complexes compared to normal platelets (P < 0.005). The quantities of plasminogen stored in platelets were important to induce QPD-like proteolysis of normal alpha-granule proteins by two chain u-PA (tcu-PA) in vitro. Moreover, adding supplemental plasminogen to QPD, but not to control, platelet lysates, triggered further alpha-granule protein proteolysis to forms that comigrated with plasmin degraded proteins. These data suggest the generation of increased but limiting amounts of plasmin within platelets is involved in producing the unique phenotypic changes to alpha-granule proteins in QPD platelets. The QPD is the only known bleeding disorder associated with chronic, intracellular activation of the fibrinolytic cascade.

Anti-invasion effects of 6-shogaol and 6-gingerol, two active components in ginger, on human hepatocarcinoma cells.

PubMed

Weng, Chia-Jui; Wu, Cheng-Feng; Huang, Hsiao-Wen; Ho, Chi-Tang; Yen, Gow-Chin

2010-11-01

Hepatocellular carcinoma is the most common type of liver cancer and is highly metastatic. Metastasis is considered to be the major cause of death in cancer patients. Ginger is a natural dietary rhizome with anti-oxidative, anti-inflammatory, and anti-carcinogenic activities. The aims of this study were to evaluate the anti-invasion activity of 6-shogaol and 6-gingerol, two compounds found in ginger, on hepatoma cells. The migratory and invasive abilities of phorbol 12-myristate 13-acetate (PMA)-treated HepG2 and PMA-untreated Hep3B cells were both reduced in a dose-dependent manner by treatment with 6-shogaol and 6-gingerol. Upon incubation of PMA-treated HepG2 cells and PMA-untreated Hep3B cells with 6-shogaol and 6-gingerol, matrix metalloproteinase (MMP)-9 activity decreased, whereas the expression of tissue inhibitor metalloproteinase protein (TIMP)-1 increased in both cell types. Additionally, urokinase-type plasminogen activator activity was dose-dependently decreased in Hep3B cells after incubation with 6-shogaol for 24 h. Analysis with semi-quantitative reverse transcription-PCR showed that the regulation of MMP-9 by 6-shogaol and 6-gingerol and the regulation of TIMP-1 by 6-shogaol in Hep3B cells may on the transcriptional level. These results suggest that 6-shogaol and 6-gingerol might both exert anti-invasive activity against hepatoma cells through regulation of MMP-9 and TIMP-1 and that 6-shogaol could further regulate urokinase-type plasminogen activity.

Prevotella intermedia stimulates tissue-type plasminogen activator and plasminogen activator inhibitor-2 expression via multiple signaling pathways in human periodontal ligament cells.

PubMed

Guan, Su-Min; He, Jian-Jun; Zhang, Ming; Shu, Lei

2011-06-01

Prevotella intermedia is an important periodontal pathogen that induces various inflammatory and immune responses. In this study, we investigated the effects of P. intermedia on the plasminogen system in human periodontal ligament (hPDL) cells and explored the signaling pathways involved. Using semi-quantitative reverse transcription (RT)-PCR and quantitative real-time RT-qPCR, we demonstrated that P. intermedia challenge increased tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)-2 expression in a concentration- and time-dependent manner, but exerted no influence on urokinase-type plasminogen activator and PAI-1mRNA expression in hPDL cells. Prevotella intermedia stimulation also enhanced tPA protein secretion as confirmed by enzyme-linked immunosorbent assay. Western blot results revealed that P. intermedia treatment increased phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase (p38). ERK, JNK and protein kinase C inhibitors significantly attenuated the P. intermedia-induced tPA and PAI-2 expression. Furthermore, p38 and phosphatidylinositol 3-kinase inhibitors markedly decreased PAI-2 expression, whereas they showed no or little inhibition on tPA expression. In contrast, inhibition of protein kinase A greatly enhanced the upregulatory effect of P. intermedia on tPA and PAI-2 expression. Our results suggest that P. intermedia may contribute to periodontal tissue destruction by upregulating tPA and PAI-2 expression in hPDL cells via multiple signaling pathways. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Molecular Disruption of Breast Tumor Angiogenesis

DTIC Science & Technology

2004-07-01

human breast cancer . During the period of this grant, we carried out studies to confirm that targeted ablation of PAI- 1 gene expression resulted in a...microvessel endothelial cells. Breast cancer -derived factors (TGF-P, EGF)were found to be important contributors of continued PAI-I expression and long...various culture model systems implicated both urokinase plasminogen activator (uPA) and its fast-acting type-i inhibitor (PAl-l) as necessary to achieve

Berberine suppresses in vitro migration and invasion of human SCC-4 tongue squamous cancer cells through the inhibitions of FAK, IKK, NF-kappaB, u-PA and MMP-2 and -9.

PubMed

Ho, Yung-Tsuan; Yang, Jai-Sing; Li, Tsai-Chung; Lin, Jen-Jyh; Lin, Jaung-Geng; Lai, Kuang-Chi; Ma, Chia-Yu; Wood, W Gibson; Chung, Jing-Gung

2009-07-08

There is increasing evidence that urokinase-type plasminogen activator (u-PA) and matrix metalloproteinases (MMPs) play an important role in cancer metastasis and angiogenesis. Inhibition of u-PA and MMPs could suppress migration and invasion of cancer cells. Berberine, one of the main constituents of the plant Rhizoma coptidis, is a type of isoquinoline alkaloid, reported to have anti-cancer effects in different human cancer cell lines. There is however, no available information on effects of berberine on migration and invasion of human tongue cancer cells. Here, we report that berberine inhibited migration and invasion of human SCC-4 tongue squamous carcinoma cells. This action was mediated by the p-JNK, p-ERK, p-p38, IkappaK and NF-kappaB signaling pathways resulting in inhibition of MMP-2 and -9 in human SCC-4 tongue squamous carcinoma cells. Our Western blowing analysis also showed that berberine inhibited the levels of urokinase-plasminogen activator (u-PA). These results suggest that berberine down-regulates u-PA, MMP-2 and -9 expressions in SCC-4 cells through the FAK, IKK and NF-kappaB mediated pathways and a novel function of berberine is to inhibit the invasive capacity of malignant cells.

Urokinase-type plasminogen activator: a new target for male contraception?

PubMed

Qin, Ying; Han, Yan; Xiong, Cheng-Liang; Li, Hong-Gang; Hu, Lian; Zhang, Ling

2015-01-01

Urokinase-type plasminogen activator (uPA) is closely related to male reproduction. With the aim of investigating the possibility for uPA as a potential contraceptive target, in the present work, Kunming male mice were immunized by human uPA subcutaneous injection at three separate doses for 3 times. Then the potency of the anti-human uPA antibody in serum was analyzed, and mouse fertility was evaluated. Serum antibody titers for human uPA in immunized groups all reached 1:10,240 or higher levels by enzyme linked immunosorbent assay, and mating experiments revealed that pregnancy rates and the mean number of embryos implanted after mating declined obviously (P < 0.05) when compared with control groups. However, the mating capacity and reproductive organ weights had no obvious change, and histological analysis of the testes and epididymides also showed normal morphology for immunized male mice. Sperm function tests suggested that the sperm concentration, sperm viability, sperm motility, and in vitro fertilization rate for the cauda epididymis sperm in uPA-immunized groups were lower than those in the controls (P < 0.05). Together, these observations indicated that subcutaneous injection human uPA to the male mice could effectively reduce their fertility, and uPA could become a new target for immunocontraception in male contraceptive development.

Effects of Heparin and ε-Aminocaproic Acid in Dogs on Plasmin- 125I Generation in Response to Urokinase Injections and Venous Injury

PubMed Central

Takeda, Y.; Parkhill, T. R.; Nakabayashi, M.

1972-01-01

The isotopic method described previously for quantification of plasmin- 125I by disc gel electrophoresis was modified by inclusion of euglobulin precipitation to expand its applicability to plasmas containing low radioactivity of plasmin- 125I and plasminogen- 125I. It was found that the euglobulin precipitation method precipitates 72.4±2.1 (sd)% of both plasmin- 125I and plasminogen- 125I. Using this method and plasminogen- 125I as a tracer, studies were first made of the effects of heparin and ε-aminocaproic acid in dogs on plasmin- 125I generation in responese to a single injection of urokinase and to venous injury; second, of the effects of venous occlusion and thrombosis on plasmin- 125I generation; and third, in vitro studies of plasminogen- 125I affinity to fibrin and its activation in blood clots. The venous injury was produced by the damage of venous endothelium by an injection of 90% phenol and the thrombosis by a thrombin injection into an occluded vein. Heparin and ε-aminocaproic acid under the present experimental conditions inhibited about 78 and 100%, respectively of plasmin- 125I generation by the urokinase injection. Similar inhibitory effects of heparin and ε-aminocaproic acid were observed on plasmin- 125I generation in response to venous injury. The venous occlusion caused a small degree of plasmin- 125I generation, but thrombin thrombosis did not seem to stimulate the generation of plasmin- 125I. The in vitro studies showed that plasminogen- 125I does not have a specific affinity to fibrin and is incorporated into blood clots in approximately equal concentrations as those in serum during clotting processes, and that blood clots per se do not stimulate plasmin- 125I generation. These results suggest that injured veins release considerable amounts of vascular plasminogen activators into circulation and that these play an important role in thrombus dissolution in vivo. PMID:4262519

Synthesis and characterization of an 111In-labeled peptide for the in vivo localization of human cancers expressing the urokinase-type plasminogen activator receptor (uPAR)

PubMed Central

Liu, Dijie; Overbey, Douglas; Watkinson, Lisa; Giblin, Michael F.

2009-01-01

This study describes the synthesis and preliminary biologic evaluation of an 111Inlabeled peptide antagonist of the urokinase-type plasminogen activator receptor (uPAR) as a potential probe for assessing metastatic potential of human breast cancer in vivo. The peptide (NAc-dD-CHA-F-dS-dR-Y-L-W-S-βAla)2-K-K(DOTA)-NH2 was synthesized and conjugated with the DOTA chelating moiety via conventional Solid-Phase Peptide Synthesis (SPPS), purified by reversed-phase HPLC, and characterized by MALDI-TOF MS and receptor binding assay. In vitro receptor binding studies demonstrated an IC50 of 240 ± 125 nM for the peptide, compared with IC50’s of 0.44 ± 0.02 and 0.75 ± 0.01 nM for the amino terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA) and full-length uPA, respectively. In vivo biodistribution studies were carried out using SCID mice bearing MDA-MB-231 human breast cancer xenografts. Biodistribution data was collected at 1, 4, and 24 hr post-injection of 111In-DOTA-peptide, and compared with data obtained using a scrambled control peptide, as well as with data obtained using wild-type ATF radiolabeled with I-125. Biodistribution studies showed rapid elimination of the 111In-labeled peptide from the blood pool, with 0.12 ± 0.06% ID/g remaining in blood at 4 hr pi. Elimination was seen primarily via the renal/urinary route, with 83.9 ± 2.2%ID in the urine at the same timepoint. Tumor uptake at this time was 0.53 ± 0.11%ID/g, resulting in tumor: blood and tumor: muscle ratios of 4.2 and 9.4, respectively. Uptake in tumor was significantly higher than that obtained using a scrambled control peptide that showed no specific binding to uPAR (p < 0.05). In vitro and ex vivo results both suggested that the magnitude of tumor-specific binding was reduced in this model by endogenous expression of uPA. The results indicate that radiolabeled peptide uPAR antagonists may find application in the imaging and therapy of uPAR-expressing breast cancers in vivo. PMID:19354275

Cadmium exposure is associated with soluble urokinase plasminogen activator receptor, a circulating marker of inflammation and future cardiovascular disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fagerberg, Björn, E-mail: bjorn.fagerberg@wlab.gu.

Background: Diet and smoking are the main sources of cadmium exposure in the general population. Cadmium increases the risk of cardiovascular diseases, and experimental studies show that it induces inflammation. Blood cadmium levels are associated with macrophages in human atherosclerotic plaques. Soluble urokinase-type plasminogen activator receptor (suPAR) is an emerging biomarker for cardiovascular events related to inflammation and atherosclerotic plaques. The aim was to examine whether blood cadmium levels are associated with circulating suPAR and other markers of inflammation. Methods: A population sample of 4648 Swedish middle-aged women and men was examined cross-sectionally in 1991–1994. Plasma suPAR was assessed bymore » ELISA, leukocytes were measured by standard methods, and blood cadmium was analysed by inductively coupled plasma mass spectrometry. Prevalent cardiovascular disease, ultrasound-assessed carotid plaque occurrence, and several possible confounding factors were recorded. Results: After full adjustment for risk factors and confounding variables, a 3-fold increase in blood cadmium was associated with an 10.9% increase in suPAR concentration (p<0.001). In never-smokers, a 3-fold increase in blood cadmium was associated with a 3.7% increase in suPAR concentration (p<0.01) after full adjustment. Blood cadmium was not associated with C-reactive protein, white blood cell count and Lp-PLA2 but with neutrophil/lymphocyte ratio in one of two statistical models. Conclusions: Exposure to cadmium was associated with increased plasma suPAR in the general population, independently of smoking and cardiovascular disease. These results imply that cadmium is a possible cause for raised levels of this inflammatory marker. - Highlights: • Cadmium is a toxic proinflammatory, proatherosclerotic metal. • Soluble urokinase-type plasminogen activator receptor (suPAR) in plasma is a promising proinflammatory marker of atherosclerosis. • Blood cadmium and plasma suPAR were measured in a cohort of 4648 Swedish men and women. • Blood cadmium was positively associated with plasma suPAR, also in never smokers.« less

Leukemia inhibitory factor promote trophoblast invasion via urokinase-type plasminogen activator receptor in preeclampsia.

PubMed

Zheng, Qin; Dai, Kuixing; Cui, Xinyuan; Yu, Ming; Yang, Xuesong; Yan, Bin; Liu, Shuai; Yan, Qiu

2016-05-01

Preeclampsia is a pregnancy-related syndrome which can cause perinatal mortality and morbidity. Inadequate invasion by trophoblast cells may lead to poor perfusion of the placenta, even result in preeclampsia. Understanding the molecular mechanisms underlying placentation facilitates the better intervention of preeclampsia. Urokinase-type plasminogen activator receptor (uPAR) is involved in the physiological and pathological processes. Leukemia inhibitory factor (LIF) is an important regulator in the establishment of pregnancy. However, the expression of uPAR in preeclamptic patients and its relationship with LIF remains unclear. In the current study, we found that the level of uPAR was relatively lower in the placentas from preeclamptic patients as compared with normal pregnant women. LIF promoted trophoblast cell outgrowth by upregulating uPAR in an explants culture, and LIF also enhanced migration and invasion potential through uPAR in trophoblast JAR and JEG-3 cell lines, and with increased gelatinolytic activities of matrix metalloproteinase 2 (MMP-2). The effect of LIF and uPAR on trophoblast migration and invasion was mediated by PI3K/AKT signaling pathway. Our data indicates the roles of LIF in promoting trophoblast migration and invasion through uPAR and suggest that abnormal expression of uPAR might be associated with the etiology of preeclampsia. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Plasmin cleaves fibrinogen and the human complement proteins C3b and C5 in the presence of Leptospira interrogans proteins: A new role of LigA and LigB in invasion and complement immune evasion.

PubMed

Castiblanco-Valencia, Mónica Marcela; Fraga, Tatiana Rodrigues; Pagotto, Ana Helena; Serrano, Solange Maria de Toledo; Abreu, Patricia Antonia Estima; Barbosa, Angela Silva; Isaac, Lourdes

2016-05-01

Plasminogen is a single-chain glycoprotein found in human plasma as the inactive precursor of plasmin. When converted to proteolytically active plasmin, plasmin(ogen) regulates both complement and coagulation cascades, thus representing an important target for pathogenic microorganisms. Leptospira interrogans binds plasminogen, which is converted to active plasmin. Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules that interact with extracellular matrix components and complement regulators, including proteins of the FH family and C4BP. In this work, we demonstrate that these multifunctional molecules also bind plasminogen through both N- and C-terminal domains. These interactions are dependent on lysine residues and are affected by ionic strength. Competition assays suggest that plasminogen does not share binding sites with C4BP or FH on Lig proteins at physiological molar ratios. Plasminogen bound to Lig proteins is converted to proteolytic active plasmin in the presence of urokinase-type plasminogen activator (uPA). Lig-bound plasmin is able to cleave the physiological substrates fibrinogen and the complement proteins C3b and C5. Taken together, our data point to a new role of LigA and LigB in leptospiral invasion and complement immune evasion. Plasmin(ogen) acquisition by these versatile proteins may contribute to Leptospira infection, favoring bacterial survival and dissemination inside the host. Copyright © 2016. Published by Elsevier GmbH.

Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes

PubMed Central

Alfano, Massimo; Cinque, Paola; Giusti, Guido; Proietti, Silvia; Nebuloni, Manuela; Danese, Silvio; D’Alessio, Silvia; Genua, Marco; Portale, Federica; Lo Porto, Manuela; Singhal, Pravin C.; Rastaldi, Maria Pia; Saleem, Moin A.; Mavilio, Domenico; Mikulak, Joanna

2015-01-01

Increased plasma level of soluble urokinase-type plasminogen activator receptor (suPAR) was associated recently with focal segmental glomerulosclerosis (FSGS). In addition, different clinical studies observed increased concentration of suPAR in various glomerular diseases and in other human pathologies with nephrotic syndromes such as HIV and Hantavirus infection, diabetes and cardiovascular disorders. Here, we show that suPAR induces nephrin down-modulation in human podocytes. This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms’ tumor 1 (WT-1) transcription factor expression. Moreover, an antagonist of αvβ3 integrin RGDfv blocked suPAR-induced suppression of nephrin. These in vitro data were confirmed in an in vivo uPAR knock out Plaur−/− mice model by demonstrating that the infusion of suPAR inhibits expression of nephrin and WT-1 in podocytes and induces proteinuria. This study unveiled that interaction of full-length suPAR with αvβ3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR. PMID:26380915

Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

PubMed

Montesinos, M Carmen; Desai-Merchant, Avani; Cronstein, Bruce N

2015-12-01

Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing.

Urokinase-Type Plasminogen Activator in a Human Sarcoma Cellular Model for Metastasis in Athymic Mice

DTIC Science & Technology

1990-05-01

essential medium, used for tissue culture DMSO- Dimemthyl sulfoxide, an inhibitor of uPA production E-ACA- Epsilon aminocaproic acid , an inhibitor of uPA...inhibitors, such as E- aminocaproic acid (E-ACA), phenylmethanesulfonyl fluoride, diisopropyl-fluorophosphate, alpha tocopherol, metal ions (especially Zn...plasma was demonstrated approximately 25 years later (Astedt et al., 1978). The complete amino acid sequence of uPA was reported in 1982 (Gunzler et

Urokinase plasminogen activator mRNA is induced by IL-1alpha and TNF-alpha in in vitro acantholysis.

PubMed

Feliciani, Claudio; Toto, Paola; Wang, Binghe; Sauder, Daniel N; Amerio, Pierluigi; Tulli, Antonio

2003-08-01

The role of urokinase type plasminogen activator (uPA) has been well documented in the pathogenesis of pemphigus vulgaris (PV). Activation of plasminogen into active serine protease plasmin initiates extracellular proteolysis leading to acantholysis but the mechanisms underlying this process are not clearly understood. We have previously shown that keratinocyte derived cytokines IL-1alpha and TNF-alpha are involved in PV-induced acantholysis. In the present study we sought to examine whether keratinocyte-derived IL-1alpha and TNF-alpha are correlated with uPA induction in keratinocytes during acantholysis. Normal human keratinocytes were incubated with diluted PV serum. mRNAs for IL-1alpha, TNF-alpha and uPA were examined with RT-PCR at various time points and acantholysis was measured. IL-1alpha, TNF-alpha and uPA mRNAs were all induced in keratinocytes following PV serum stimulation; IL-1alpha/TNF-alpha mRNAs' expression was earlier than the expression of uPA mRNA. To further examine the role of IL-1alpha, TNF-alpha and uPA in acantholysis, we performed antibody blocking studies. Anti-IL-1alpha, anti-TNF-alpha and anti-uPA antibodies suppressed acantholysis by 76%, 80% and 90%, respectively. In addition, anti-IL-1alpha and anti-TNF-alpha antibodies inhibited uPA mRNA induction, whereas anti-uPA antibodies did not alter IL-1alpha/TNF-alpha mRNAs' expression. Our results confirm the role of uPA in acantholysis and suggest an involvement of IL-1alpha/TNF-alpha in uPA induction.

Downregulation of Extracellular Matrix Metalloproteinase Inducer by scFv-M6-1B9 Intrabody Suppresses Cervical Cancer Invasion Through Inhibition of Urokinase-Type Plasminogen Activator.

PubMed

Panich, Tipattaraporn; Tragoolpua, Khajornsak; Pata, Supansa; Tayapiwatana, Chatchai; Intasai, Nutjeera

2017-02-01

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN) accelerates tumor invasion and metastasis via activation of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) expression. The authors were interested in whether the scFv-M6-1B9 intrabody against EMMPRIN that retains EMMPRIN in endoplasmic reticulum could be a potential tool to suppress cervical cancer invasion through inhibition of uPA. The chimeric adenoviral vector Ad5/F35-scFv-M6-1B9 was transferred into human cervical carcinoma HeLa cells to produce the scFv-M6-1B9 intrabody against EMMPRIN. Cell surface expression of EMMPRIN, the membrane-bound uPA, the enzymatic activity of secreted uPA, and the invasion ability were analyzed. The scFv-M6-1B9 intrabody successfully diminished the cell surface expression of EMMPRIN and the membrane-bound uPA on HeLa cells. uPA activity from tissue culture media of EMMPRIN-downregulated HeLa cells was decreased. The invasion ability of HeLa cells harboring scFv-M6-1B9 intrabody was also suppressed. These results suggested that the scFv-M6-1B9 intrabody might represent a potential approach for invasive cervical cancer treatment. The application of scFv-M6-1B9 intrabody in animal experiments and preclinical studies would be investigated further.

Small molecule antagonists of the urokinase (uPA): urokinase receptor (uPAR) interaction with high reported potencies show only weak effects in cell-based competition assays employing the native uPAR ligand.

PubMed

De Souza, Melissa; Matthews, Hayden; Lee, Jodi A; Ranson, Marie; Kelso, Michael J

2011-04-15

Binding of the urokinase-type plasminogen activator (uPA) to its cell-surface-bound receptor uPAR and upregulation of the plasminogen activation system (PAS) correlates with increased metastasis and poor prognosis in several tumour types. Disruptors of the uPA:uPAR interaction represent promising anti-tumour/metastasis agents and several approaches have been explored for this purpose, including the use of small molecule antagonists. Two highly potent non-peptidic antagonists 1 and 2 (IC(50)1=0.8 nM, IC(50)2=33 nM) from the patent literature were reportedly identified using competition assays employing radiolabelled uPAR-binding uPA fragments and appeared as useful pharmacological tools for studying the PAS. Before proceeding to such studies, confirmation was sought that 1 and 2 retained their potencies in physiologically relevant cell-based competition assays employing uPAR's native binding partner high molecular weight uPA (HMW-uPA). This study describes a new solution phase synthesis of 1, a mixed solid/solution phase synthesis of 2 and reports the activities of 1 and 2 in semi-quantitative competition flow cytometry assays and quantitative cell-based uPA activity assays that employed HMW-uPA as the competing ligand. The flow cytometry experiments revealed that high concentrations of 2 (10-100 μM) are required to compete with HMW-uPA for uPAR binding and that 1 shows no antagonist effects at 100 μM. The cell-based enzyme activity assays similarly revealed that 1 and 2 are poor inhibitors of cell surface-bound HMW-uPA activity (IC(50) >100 μM for 1 and 2). The report highlights the dangers of identifying false-positive lead uPAR antagonists from competition assays employing labelled competing ligands other than the native HMW-uPA. Copyright © 2011 Elsevier Ltd. All rights reserved.

Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation

PubMed Central

Veljkovic, D. Kika; Rivard, Georges E.; Diamandis, Maria; Blavignac, Jessica; Cramer-Bordé, Elisabeth M.

2009-01-01

Quebec platelet disorder (QPD) is an inherited bleeding disorder associated with increased urokinase plasminogen activator (uPA) in platelets but not in plasma, intraplatelet plasmin generation, and α-granule protein degradation. These abnormalities led us to investigate uPA expression by QPD CD34+ progenitors, cultured megakaryocytes, and platelets, and whether uPA was stored in QPD α-granules. Although QPD CD34+ progenitors expressed normal amounts of uPA, their differentiation into megakaryocytes abnormally increased expression of the uPA gene but not the flanking genes for vinculin or calcium/calmodulin-dependent protein kinase IIγ on chromosome 10. The increased uPA production by cultured QPD megakaryocytes mirrored their production of α-granule proteins, which was normal. uPA was localized to QPD α-granules and it showed extensive colocalization with α-granule proteins in both cultured QPD megakaryocytes and platelets, and with plasminogen in QPD platelets. In QPD megakaryocytes, cultured without or with plasma as a source of plasminogen, α-granule proteins were stored undegraded and this was associated with much less uPA-plasminogen colocalization than in QPD platelets. Our studies indicate that the overexpression of uPA in QPD emerges with megakaryocyte differentiation, without altering the expression of flanking genes, and that uPA is costored with α-granule proteins prior to their proteolysis in QPD. PMID:19029443

Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation.

PubMed

Veljkovic, D Kika; Rivard, Georges E; Diamandis, Maria; Blavignac, Jessica; Cramer-Bordé, Elisabeth M; Hayward, Catherine P M

2009-02-12

Quebec platelet disorder (QPD) is an inherited bleeding disorder associated with increased urokinase plasminogen activator (uPA) in platelets but not in plasma, intraplatelet plasmin generation, and alpha-granule protein degradation. These abnormalities led us to investigate uPA expression by QPD CD34(+) progenitors, cultured megakaryocytes, and platelets, and whether uPA was stored in QPD alpha-granules. Although QPD CD34(+) progenitors expressed normal amounts of uPA, their differentiation into megakaryocytes abnormally increased expression of the uPA gene but not the flanking genes for vinculin or calcium/calmodulin-dependent protein kinase IIgamma on chromosome 10. The increased uPA production by cultured QPD megakaryocytes mirrored their production of alpha-granule proteins, which was normal. uPA was localized to QPD alpha-granules and it showed extensive colocalization with alpha-granule proteins in both cultured QPD megakaryocytes and platelets, and with plasminogen in QPD platelets. In QPD megakaryocytes, cultured without or with plasma as a source of plasminogen, alpha-granule proteins were stored undegraded and this was associated with much less uPA-plasminogen colocalization than in QPD platelets. Our studies indicate that the overexpression of uPA in QPD emerges with megakaryocyte differentiation, without altering the expression of flanking genes, and that uPA is costored with alpha-granule proteins prior to their proteolysis in QPD.

Clinical utility of level-of-evidence-1 disease forecast cancer biomarkers uPA and its inhibitor PAI-1.

PubMed

Schmitt, Manfred; Mengele, Karin; Napieralski, Rudolf; Magdolen, Viktor; Reuning, Ute; Gkazepis, Apostolos; Sweep, Fred; Brünner, Nils; Foekens, John; Harbeck, Nadia

2010-11-01

The prognostic and/or predictive value of the cancer biomarkers, urokinase-type plasminogen activator (uPA) and its inhibitor (plasminogen activator inhibitor [PAI]-1), determined by ELISA in tumor-tissue extracts, was demonstrated for several cancer types in numerous clinically relevant retrospective or prospective studies, including a multicenter breast cancer therapy trial (Chemo-N0). Consequently, for the first time ever for any cancer biomarker for breast cancer, uPA and PAI-1 have reached the highest level of evidence, level-of-evidence-1. At present, two other breast cancer therapy trials, NNBC-3 and Plan B, also incorporating uPA and PAI-1 as treatment-assignment tools are in effect. Furthermore, small synthetic molecules targeting uPA are currently in Phase II clinical trials in patients afflicted with advanced cancer of the ovary, breast or pancreas.

Seahorse-derived peptide suppresses invasive migration of HT1080 fibrosarcoma cells by competing with intracellular α-enolase for plasminogen binding and inhibiting uPA-mediated activation of plasminogen.

PubMed

Kim, Yong-Tae; Kim, Se-kwon; Jeon, You-Jin; Park, Sun Joo

2014-12-01

α-Enolase is a glycolytic enzyme and a surface receptor for plasminogen. α-Enolase-bound plasminogen promotes tumor cell invasion and cancer metastasis by activating plasmin and consequently degrading the extracellular matrix degradation. Therefore, α-enolase and plasminogen are novel targets for cancer therapy. We found that the amino acid sequence of a peptide purified from enzymatic hydrolysates of seahorse has striking similarities to that of α-enolase. In this study, we report that this peptide competes with cellular α-enolase for plasminogen binding and suppresses urokinase plasminogen activator (uPA)-mediated activation of plasminogen, which results in decreased invasive migration of HT1080 fibrosarcoma cells. In addition, the peptide treatment decreased the expression levels of uPA compared to that of untreated controls. These results provide new insight into the mechanism by which the seahorse-derived peptide suppresses invasive properties of human cancer cells. Our findings suggest that this peptide could emerge as a potential therapeutic agent for cancer.

Seahorse-derived peptide suppresses invasive migration of HT1080 fibrosarcoma cells by competing with intracellular α-enolase for plasminogen binding and inhibiting uPA-mediated activation of plasminogen

PubMed Central

Kim, Yong-Tae; Kim, Se-kwon; Jeon, You-Jin; Park, Sun Joo

2014-01-01

α-Enolase is a glycolytic enzyme and a surface receptor for plasminogen. α-Enolase-bound plasminogen promotes tumor cell invasion and cancer metastasis by activating plasmin and consequently degrading the extracellular matrix degradation. Therefore, α-enolase and plasminogen are novel targets for cancer therapy. We found that the amino acid sequence of a peptide purified from enzymatic hydrolysates of seahorse has striking similarities to that of α-enolase. In this study, we report that this peptide competes with cellular α-enolase for plasminogen binding and suppresses urokinase plasminogen activator (uPA)-mediated activation of plasminogen, which results in decreased invasive migration of HT1080 fibrosarcoma cells. In addition, the peptide treatment decreased the expression levels of uPA compared to that of untreated controls. These results provide new insight into the mechanism by which the seahorse-derived peptide suppresses invasive properties of human cancer cells. Our findings suggest that this peptide could emerge as a potential therapeutic agent for cancer. [BMB Reports 2014; 47(12): 691-696] PMID:24602611

Urokinase-type Plasminogen Activator Resulting from Endometrial Carcinogenesis Enhances Tumor Invasion and Correlates with Poor Outcome of Endometrial Carcinoma Patients

PubMed Central

Huang, Chia-Yen; Chang, Ming-Cheng; Huang, Wei-Yun; Huang, Ching-Ting; Tang, Yu-Chien; Huang, Hsien-Da; Kuo, Kuan-Ting; Chen, Chi-An; Cheng, Wen-Fang

2015-01-01

The purpose of this study was to identify the dysregulated genes involved in the tumorigenesis and progression of endometrial endometrioid adenocarcinoma (EEC), and their possible mechanisms. Endometrial specimens including normal endometrial tissues, atypical endometrial hyperplasia, and EEC were analyzed. The expression profiles were compared using GeneChip Array. The gene expression levels were determined by real-time RT-PCR in the training and testing sets to correlate the clinico-pathological parameters of EEC. Immunoblotting, in vitro cell migration and invasion assays were performed in human endometrial cancer cell lines and their transfectants. In microarray analysis, seven dysregulated genes were identified. Only the levels of urokinase-type plasminogen activator (uPA) were higher in EEC with deep myometrial invasion, positive lympho-vascular space invasion, lymph node metastasis, and advanced stages. After multivariate analysis, uPA was the only independent poor prognostic factor for disease-free survival in the EEC patients (hazard ratio: 4.65, p = 0.03). uPA may enhance the migratory and invasive capabilities of endometrial tumor cells by the phosphorylation of ERK1/2, Akt and p38 molecules. uPA is a dysregulated gene involved in the tumorigenesis, bio-pathological features and outcomes of EEC. uPA may be a potential molecule and target for the detection and treatment of EEC. PMID:26033187

Bleeding risks associated with inheritance of the Quebec platelet disorder.

PubMed

McKay, Heather; Derome, Francine; Haq, M Anwar; Whittaker, Susan; Arnold, Emmy; Adam, Frédéric; Heddle, Nancy M; Rivard, Georges E; Hayward, Catherine P M

2004-07-01

Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder associated with increased urokinase-type plasminogen activator in platelets and alpha-granule protein degradation. To determine bleeding risks and common manifestations of QPD, a history questionnaire was developed and administered to 127 relatives in a family with QPD. Data entry was done blinded to affected and unaffected status, determined by assays for platelet urokinase-type plasminogen activator (u-PA) and fibrinogen degradation. Odds ratios (ORs), with 95% confidence intervals (CIs), were determined for items queried. Summative bleeding scores for each individual were calculated using items with OR more than 1. Mean ages (34 years; range, 1-89 years) were similar for affected (n = 23) and unaffected (n = 104) family members. Affected individuals had higher mean bleeding scores (P <.0001) and a much higher likelihood (OR > 20) of having bleeding that led to lifestyle changes, bruises that spread lower or as large or larger than an orange or both, joint bleeds, bleeding longer than 24 hours after dental extractions or deep cuts, and received or been recommended other treatments (fibrinolytic inhibitors) for bleeding. Individuals with QPD and exposure(s) to hemostatic challenges had experienced excessive bleeding only when fibrinolytic inhibitors had not been used. These data illustrate that QPD is associated with increased risks of bleeding that can be modified by fibrinolytic inhibitors.

Cytokine and estrogen stimulation of endothelial cells augments activation of the prekallikrein-high molecular weight kininogen complex: Implications for hereditary angioedema.

PubMed

Joseph, Kusumam; Tholanikunnel, Baby G; Kaplan, Allen P

2017-07-01

When the prekallikrein-high molecular weight kininogen complex is bound to endothelial cells, prekallikrein is stoichiometrically converted to kallikrein because of release of heat shock protein-90 (Hsp90). Although bradykinin formation is typically initiated by factor XII autoactivation, it is also possible to activate factor XII either by kallikrein, thus formed, or by plasmin. Because attacks of hereditary angioedema can be related to infection and/or exposure to estrogen, we questioned whether estrogen or cytokine stimulation of endothelial cells could augment release of Hsp90 and prekallikrein activation. We also tested release of profibrinolytic enzymes, urokinase, and tissue plasminogen activator (TPA) as a source for plasmin formation. Cells were stimulated with agonists, and secretion of Hsp90, urokinase, and TPA was measured in the culture supernatants by ELISA. Activation of the prekallikrein-HK complex was measured by using pro-phe-arg-p-nitroanilide reflecting kallikrein formation. Hsp90 release was stimulated with optimal doses of estradiol, IL-1, and TNF-α (10 ng/mL) from 15 minutes to 120 minutes. TPA release was not augmented by any of the agonists tested but urokinase was released by IL-1, TNF-α, and thrombin (positive control), but not estrogen. Augmented activation of the prekallikrein-HK complex to generate kallikrein was seen with each agonist that releases Hsp90. Addition of 0.1% factor XII relative to prekallikrein-HK leads to rapid formation of kallikrein; factor XII alone does not autoactivate. IL-1, TNF-α, and estrogen stimulate release of Hsp90 and augment activation of the prekallikrein-HK complex to generate kallikrein and bradykinin. IL-1 and TNF-α stimulate release of urokinase, which can convert plasminogen to plasmin and represents a possible source for plasmin generation in all types of hereditary angioedema, but particularly hereditary angioedema with normal C1 inhibitor with a factor XII mutation. Both kallikrein and plasmin activate factor XII; kallikrein is 20 times more potent on a molar basis. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Gene polymorphisms of fibrinolytic enzymes in coal workers' pneumoconiosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, L.C.; Tseng, J.C.; Hua, C.C.

2006-03-15

The authors assessed the gene polymorphisms of missense C/T polymorphism in exon 6 of the urokinase-plasminogen activator (PLAU) gene (PLAU P141L), A/u-repeat in intron 8 of the tissue-type plasminogen activator (PLAT) gene (PLAT TPA25 Alu insertion), and 4G/5G in the promoter region of the serine proteinase inhibitor, clade E (SERPINE) or plasminogen activator inhibitor type 1 gene (SERPINE1 -675 4G/5G) in 153 healthy volunteers and 154 retired coal miners with coal miners' pneumoconiosis (CWP). The CWP subjects included 94 individuals with simple pneumoconiosis and 60 individuals with progressive massive fibrosis presenting with worse pulmonary function. The distributions of genotypes ofmore » these three genes did not differ between the control and CWP subjects or between subjects with simple pneumoconiosis and those with progressive massive fibrosis. However, by assessing duration of work and its interaction with genotypes by means of logistic regression, the authors found the missense C/T polymorphism in exon 6 of the PLAU gene to be an effect modifier of the association between work duration and the development of progressive massive fibrosis.« less

The Role of Osteopontin in the Malignancy of Human Breast Carcinoma

DTIC Science & Technology

1998-06-01

1997): The urokinase-type plasminogen activator system in cancer metastasis: a review. Int. J. Cancer 72:1-22. 59. Yebra M, Parry GCN, Str6mblad S...involved in the motility response) (Andreasen et al., 1997; Yebra et al., 1996). The finding that human breast epithelial cells upregulated for OPN...247-255. Xuan JW, Hota C, Shigeyama Y, D’Errico JA, Somerman MJ, and Chambers AF. (1995). J. Cell Biochem., 57, 680-690. Yebra M, Parry GCN

Anti-Urokinase Receptor Antisense Oligonucleotide (uPAR-aODN) to Prevent and Cure Long-Term Space Exploration-Related Retinal Pathological Angiogenesis

NASA Astrophysics Data System (ADS)

Lazzarano, Stefano; Lulli, Matteo; Fibbi, Gabriella; Margheri, Francesca; Papucci, Laura; Serrati, Simona; Witort, Ewa; Chilla, Anastasia; Lapucci, Andrea; Donnini, Martino; Quaglierini, Paolo; Romiti, Alice; Specogna, Rebecca; Del Rosso, Mario; Capaccioli, Sergio

2008-06-01

Angiogenesis underlies a variety of physiological processes and its possible deregulation during long term space exploration needs to be investigated. Angiogenesis is a multistep process of new blood capillary formation, where degradation of the extracellular matrix (ECM) by proteolytic enzymes, including uPA (urokinase plasminogen activator) and opening the way to migration of endothelial cells (EC), is critical. Plasminogen activation system regulates angiogenesis by both uPA-driven ECM degradation and uPA receptor (uPAR). Microgravity and low dose irradiations promote tissue neoangiogeenesis and neovascularization is often common occurence in ophthalmologic pathologies. We have designed and patented the uPAR antisense oligonucleotide (aODN) and evaluated its antiangiogenetic activity by EC cellular migration and capillary morphogenesis assays. The uPAR aODN treatment caused a 75% inhibition of human microvascular EC migration and a complete inhibition of capillary morphogenesis, suggesting its therapeutic application to prevent neoangiogenesis-related ophthalmologic pathologies during space exploration.

Effect of reoxygenation on the hypoxia-induced up-regulation of serine protease inhibitor PAI-1 in head and neck cancer cells.

PubMed

Sprague, Lisa D; Mengele, Karin; Schilling, Daniela; Geurts-Moespot, Anneke; Sweep, Fred C G J; Stadler, Peter; Schmitt, Manfred; Molls, Michael

2006-01-01

In squamous cell carcinoma of the head and neck (SCCHN), hypoxia is considered a crucial physiological modulator for malignant progression, wherebythe plasminogen activation system is involved in overlapping functions such as moulding of the extracellular matrix, cell proliferation and signal transduction. Little is known about the effects of reoxygenation on the plasminogen activation system in SCCHN cells. Three human SCCHN cell lines (BHY, CAL27, FaDu) and a non-transformed human fibroblast cell line (VH7) were exposed to hypoxic (<0.5% O(2)) conditions for up to 72 h and subsequently reoxygenated at normoxic conditions for 24 h. Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) protein concentration and former protein activity were determined by ELISA and complex ELISA, respectively. Reoxygenation induced significant changes in cell-associated and secreted PAI-1 protein compared to the normoxic control. Significant increase in cell-associated and secreted uPA protein after reoxygenation was only observed for some of the cell lines. Determination of uPA-PAI-1 complex formation revealed the release of active protein into the cell supernatant. The beneficial role of reoxygenation during radiation therapy is widely accepted. However, reoxygenation does not seem to counteract the effects induced by hypoxia on the plasminogen activation system. Fatally irradiated reoxygenat- ed tumour cells might still produce sufficient amounts of 'harmful' protein and thus initiate a path for invasion and metastasis for surviving tumour cells.

Inhibitory effect of amiloride on the urokinase plasminogen activators in prostatic cancer.

PubMed

Ray, P; Bhatti, R; Gadarowski, J; Bell, N; Nasruddin, S

1998-01-01

The diuretic drug amiloride (AMLD), which competitively inhibits the catalytic activity of urokinase plasminogen activators (UPA), was used to study its effects on the proteolytic enzymes implicated in the invasiveness and metastases in a prostatic tumor model carrying two different sublines of adenocarcinoma of the prostate. Our data showed that UPA activity was significantly higher, both in the cytosol and pellet of R3327-AT3, a fast-growing highly metastatic and androgen-insensitive tumor, as compared to the G3327-G subline, a slow-growing nonmetastatic tumor of the prostate. The UPA activity in AT3 tumor dropped when the rats were treated with AMLD for 3 weeks. The UPA activity in the sera and tumor effusions from rats with AT3 tumor was significantly higher as compared to those with G subline tumor. The number of pulmonary metastatic foci was the same in untreated rats as compared to those treated with AMLD. The lymph node inspection after 3 weeks revealed no secondary tumor in the AMLD-treated group. The role of UPA in the metastases of prostate cancer is discussed.

Serine proteases in rodent hippocampus.

PubMed

Davies, B J; Pickard, B S; Steel, M; Morris, R G; Lathe, R

1998-09-04

Brain serine proteases are implicated in developmental processes, synaptic plasticity, and in disorders including Alzheimer's disease. The spectrum of the major enzymes expressed in brain has not been established previously. We now present a systematic study of the serine proteases expressed in adult rat and mouse hippocampus. Using a combination of techniques including polymerase chain reaction amplification and Northern blotting we show that tissue-type plasminogen activator (t-PA) is the major species represented. Unexpectedly, the next most abundant species were RNK-Met-1, a lymphocyte protease not reported previously in brain, and two new family members, BSP1 (brain serine protease 1) and BSP2. We report full-length sequences of the two new proteases; homologies indicate that these are of tryptic specificity. Although BSP2 is expressed in several brain regions, BSP1 expression is strikingly restricted to hippocampus. Other enzymes represented, but at lower levels, included elastase IV, proteinase 3, complement C2, chymotrypsin B, chymotrypsin-like protein, and Hageman factor. Although thrombin and urokinase-type plasminogen activator were not detected in the primary screen, low level expression was confirmed using specific polymerase chain reaction primers. In contrast, and despite robust expression of t-PA, the usual t-PA substrate plasminogen was not expressed at detectable levels.

Soluble urokinase-type plasminogen activator receptor (suPAR) and interleukin-6 levels in hyperemesis gravidarum.

PubMed

Desdicioglu, Raziye; Yildirim, Melahat; Kocaoglu, Gulcan; Demir Cendek, Busra; Avcioglu, Gamze; Tas, Emre Erdem; Sengul, Ozlem; Erel, Ozcan; Yavuz, Ayse Filiz

2017-10-09

The aim was to compare serum soluble urokinase-type plasminogen activator receptor (suPAR) levels as well as interleukin-6 levels (IL-6) in pregnant women with hyperemesis gravidarum (HG) and asymptomatic pregnant women. Our study population consists of voluntary first trimester-pregnant women who applied to the outpatient clinic of the department of obstetrics and gynecology of Ankara Ataturk Training and Research Hospital. Between February and May 2016, 60 pregnant women were included in our prospective study. Serum suPAR and IL-6 levels were evaluated with the ELISA method. Twenty-nine pregnant women with HG and 31 asymptomatic pregnant women were included in the study. Serum suPAR level in the HG group was measured as 0.36 ± 0.56 ng/ml, whereas this level in the healthy pregnant control group was measured as 0.15 ± 0.15 ng/ml (p < 0.05). The interleukin-6 level in the HG group was 5.69 ± 2.16 pg/ml, whereas in the control group it was measured as 3.88 ± 0.28 pg/ml (p < 0.05). Serum suPAR and IL-6 levels proved to be high in the HG group. It is likely that suPAR could play a role in the etiopathogenesis of hyperemesis gravidarum. Copyright © 2017. Published by Elsevier Taiwan LLC.

Circulating intact and cleaved forms of the urokinase-type plasminogen activator receptor: biological variation, reference intervals and clinical useful cut-points.

PubMed

Thurison, Tine; Christensen, Ib J; Lund, Ida K; Nielsen, Hans J; Høyer-Hansen, Gunilla

2015-01-15

High levels of circulating forms of the urokinase-type plasminogen activator receptor (uPAR) are significantly associated to poor prognosis in cancer patients. Our aim was to determine biological variations and reference intervals of the uPAR forms in blood, and in addition, to test the clinical relevance of using these as cut-points in colorectal cancer (CRC) prognosis. uPAR forms were measured in citrated and EDTA plasma samples using time-resolved fluorescence immunoassays. Diurnal, intra- and inter-individual variations were assessed in plasma samples from cohorts of healthy individuals. Reference intervals were determined in plasma from healthy individuals randomly selected from a Danish multi-center cross-sectional study. A cohort of CRC patients was selected from the same cross-sectional study. The reference intervals showed a slight increase with age and women had ~20% higher levels. The intra- and inter-individual variations were ~10% and ~20-30%, respectively and the measured levels of the uPAR forms were within the determined 95% reference intervals. No diurnal variation was found. Applying the normal upper limit of the reference intervals as cut-point for dichotomizing CRC patients revealed significantly decreased overall survival of patients with levels above this cut-point of any uPAR form. The reference intervals for the different uPAR forms are valid and the upper normal limits are clinically relevant cut-points for CRC prognosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity

PubMed Central

Lionikiene, Ausra S.; Georgiev, Georgi; Klemmer, Anja; Brain, Chelsea; Kim, Paul Y.

2016-01-01

Activated factor XII (FXIIa) has plasminogen activator capacity but its relative contribution to fibrinolysis is considered marginal compared with urokinase and tissue plasminogen activator. Polyphosphate (polyP) is released from activated platelets and mediates FXII activation. Here, we investigate the contribution of polyP to the plasminogen activator function of αFXIIa. We show that both polyP70, of the chain length found in platelets (60-100 mer), and platelet-derived polyP significantly augment the plasminogen activation capacity of αFXIIa. PolyP70 stimulated the autoactivation of FXII and subsequent plasminogen activation, indicating that once activated, αFXIIa remains bound to polyP70. Indeed, complex formation between polyP70 and αFXIIa provides protection against autodegradation. Plasminogen activation by βFXIIa was minimal and not enhanced by polyP70, highlighting the importance of the anion binding site. PolyP70 did not modulate plasmin activity but stimulated activation of Glu and Lys forms of plasminogen by αFXIIa. Accordingly, polyP70 was found to bind to FXII, αFXIIa, and plasminogen, but not βFXIIa. Fibrin and polyP70 acted synergistically to enhance αFXIIa-mediated plasminogen activation. The plasminogen activator activity of the αFXIIa-polyP70 complex was modulated by C1 inhibitor and histidine-rich glycoprotein, but not plasminogen activator inhibitors 1 and 2. Platelet polyP and FXII were found to colocalize on the activated platelet membrane in a fibrin-dependent manner and decorated fibrin strands extending from platelet aggregates. We show that in the presence of platelet polyP and the downstream substrate fibrin, αFXIIa is a highly efficient and favorable plasminogen activator. Our data are the first to document a profibrinolytic function of platelet polyP. PMID:27694320

Spin filter perfusion system for high density cell culture: production of recombinant urinary type plasminogen activator in CHO cells.

PubMed

Avgerinos, G C; Drapeau, D; Socolow, J S; Mao, J I; Hsiao, K; Broeze, R J

1990-01-01

We have used a 20 liter stirred tank fermentor, equipped with a 127 mesh ethylene-tetrafluoroethylene rotating screen for cell recycle, for the continuous production of recombinant single chain urokinase-type plasminogen activator (rscu-PA) from Chinese hamster ovary (CHO) cells. Viable cell densities between 60 and 74 million per ml were maintained at medium perfusion rates of 3.0 to 4.0 fermentor volumes per day. Cells were retained by the 120 micron nominal opening filter through the formation of "clumped" cell aggregates of 200 to 600 microns in size, which did not foul the filter. In 31 days of culture, a total of 51 grams of rscu-PA were produced in 1,000 liters of medium. The rscu-PA produced over the course of this continuous culture was purified and characterized both in vitro and in vivo and shown to be comparable to natural scu-PA produced from the transformed human kidney cell line, TCL-598.

Plasminogen associates with phosphatidylserine-exposing platelets and contributes to thrombus lysis under flow

PubMed Central

Whyte, Claire S.; Swieringa, Frauke; Mastenbroek, Tom G.; Lionikiene, Ausra S.; Lancé, Marcus D.; van der Meijden, Paola E. J.; Heemskerk, Johan W. M.

2015-01-01

The interaction of plasminogen with platelets and their localization during thrombus formation and fibrinolysis under flow are not defined. Using a novel model of whole blood thrombi, formed under flow, we examine dose-dependent fibrinolysis using fluorescence microscopy. Fibrinolysis was dependent upon flow and the balance between fibrin formation and plasminogen activation, with tissue plasminogen activator-mediated lysis being more efficient than urokinase plasminogen activator-mediated lysis. Fluorescently labeled plasminogen radiates from platelet aggregates at the base of thrombi, primarily in association with fibrin. Hirudin attenuates, but does not abolish plasminogen binding, denoting the importance of fibrin. Flow cytometry revealed that stimulation of platelets with thrombin/convulxin significantly increased the plasminogen signal associated with phosphatidylserine (PS)-exposing platelets. Binding was attenuated by tirofiban and Gly-Pro-Arg-Pro amide, confirming a role for fibrin in amplifying plasminogen binding to PS-exposing platelets. Confocal microscopy revealed direct binding of plasminogen and fibrinogen to different platelet subpopulations. Binding of plasminogen and fibrinogen co-localized with PAC-1 in the center of spread platelets. In contrast, PS-exposing platelets were PAC-1 negative, and bound plasminogen and fibrinogen in a protruding “cap.” These data show that different subpopulations of platelets harbor plasminogen by diverse mechanisms and provide an essential scaffold for the accumulation of fibrinolytic proteins that mediate fibrinolysis under flow. PMID:25712989

Inhibition of urokinase-type plasminogen activator expression by dihydroartemisinin in breast cancer cells

PubMed Central

ZHANG, SHUQUN; MA, YINAN; JIANG, JIANTAO; DAI, ZHIJUN; GAO, XIAOYAN; YIN, XIAORAN; XI, WENTAO; MIN, WEILI

2014-01-01

The aim of the present study was to investigate the inhibitory effects of dihydroartemisinin (DHA) on the primary tumor growth and metastasis of the human breast cancer cell line, MDA-MB-231, in vitro. The expression levels of urokinase-type plasminogen activator (uPA) were detected by immunocytochemistry in two cell lines (MCF-7 and MDA-MB-231). The MDA-MB-231 cell activity was inhibited by various concentration gradients of DHA. The inhibitory rate, cell growth curve and apoptotic morphological observations were obtained using the MTT assay at 0, 24, 48 and 72 h. Cell scratch migration was performed at various time-points to test the cell proliferation and migration capacity. Reverse transcription-polymerase chain reaction was used to analyze the effect of DHA on uPA mRNA expression in breast cancer cells. The human breast cancer cell line, MDA-MB-231, possesses higher metastatic potential and relatively higher expression of uPA when compared with the MCF-7 cell line. DHA was found to inhibit the proliferation and migration capacity of the cell line, MDA-MB-231, in vitro. The growth inhibition occurred in a time- and dose-dependent manner, with IC50 values of 117.76±0.04, 60.26±0.12 and 52.96±0.07 μmol/l following 24, 48 and 72 h, respectively. The inhibition of uPA was observed to decrease breast cancer cell growth and migration. Thus, results of the present study indicate that DHA may be used for further studies with regard to breast cancer therapy. PMID:24765140

Post-Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor Expression in Lipopolysaccharide-induced Acute Lung Injury

PubMed Central

Bhandary, Yashodhar P.; Velusamy, Thirunavukkarasu; Shetty, Praveenkumar; Shetty, Rashmi S.; Idell, Steven; Cines, Douglas B.; Jain, Deepika; Bdeir, Khalil; Abraham, Edward; Tsuruta, Yuko; Shetty, Sreerama

2009-01-01

Rationale: Urokinase-type plasminogen activator (uPA) receptor (uPAR) is required for the recruitment of neutrophils in response to infection. uPA induces its own expression in lung epithelial cells, which involves its interaction with cell surface uPAR. Regulation of uPAR expression is therefore crucial for uPA-mediated signaling in infectious acute lung injury (ALI). Objectives: To determine the role of uPA in uPAR expression during ALI caused by sepsis. Methods: We used Western blot, Northern blot, Northwestern assay, and immunohistochemistry. Phosphate-buffered saline– and lipopolysaccharide (LPS)-treated wild-type and uPA−/− mice were used. Measurements and Main Results: Biological activities of uPA, including proteolysis, cell adhesion, migration, proliferation, and differentiation, are dependent on its association with uPAR. Bacterial endotoxin (LPS) is a major cause of pulmonary dysfunction and infection-associated mortality. The present study shows that LPS induces uPAR expression both in vitro and in vivo, and that the mechanism involves post-transcriptional stabilization of uPAR mRNA by reciprocal interaction of phosphoglycerate kinase (PGK) and heterogeneous nuclear ribonucleoprotein C (hnRNPC) with uPAR mRNA coding region and 3′ untranslated region determinants, respectively. The process involves tyrosine phosphorylation of PGK and hnRNPC. uPA−/− mice failed to induce uPAR expression after LPS treatment. In these mice, LPS treatment failed to alter the binding of PGK and hnRNPC protein with uPAR mRNA due to lack of tyrosine phosphorylation. Conclusions: Our study shows that induction of LPS-mediated uPAR expression is mediated through tyrosine phosphorylation of PGK and hnRNPC. This involves expression of uPA as an obligate intermediary. PMID:19029002

Plasminogen activation independent of uPA and tPA maintains wound healing in gene-deficient mice

PubMed Central

Lund, Leif R; Green, Kirsty A; Stoop, Allart A; Ploug, Michael; Almholt, Kasper; Lilla, Jennifer; Nielsen, Boye S; Christensen, Ib J; Craik, Charles S; Werb, Zena; Danø, Keld; Rømer, John

2006-01-01

Simultaneous ablation of the two known activators of plasminogen (Plg), urokinase-type (uPA) and the tissue-type (tPA), results in a substantial delay in skin wound healing. However, wound closure and epidermal re-epithelialization are significantly less impaired in uPA;tPA double-deficient mice than in Plg-deficient mice. Skin wounds in uPA;tPA-deficient mice treated with the broad-spectrum matrix metalloproteinase (MMP) inhibitor galardin (N-[(2R)-2-(hydroxamido-carbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) eventually heal, whereas skin wounds in galardin-treated Plg-deficient mice do not heal. Furthermore, plasmin is biochemically detectable in wound extracts from uPA;tPA double-deficient mice. In vivo administration of a plasma kallikrein (pKal)-selective form of the serine protease inhibitor ecotin exacerbates the healing impairment of uPA;tPA double-deficient wounds to a degree indistinguishable from that observed in Plg-deficient mice, and completely blocks the activity of pKal, but not uPA and tPA in wound extracts. These findings demonstrate that an additional plasminogen activator provides sufficient plasmin activity to sustain the healing process albeit at decreased speed in the absence of uPA, tPA and galardin-sensitive MMPs and suggest that pKal plays a role in plasmin generation. PMID:16763560

Effects of gestational hypertension and pre-eclampsia in mRNA expression of fibrinolysis genes in primary cultured human umbilical vein endothelial cells.

PubMed

Poblete-Naredo, Irais; Rodríguez-Yáñez, Yury; Corona-Núñez, Rogelio O; González-Monroy, Stuart; Salinas, Juan E; Albores, Arnulfo

2018-05-17

Hypertension disorders (HD) and pre-eclampsia (PRE) are leading causes of maternal deaths worldwide. PRE is associated with vascular endothelial dysfunction and with deregulation of the fibrinolysis pathway genes. Fibrinolysis is the fibrin clot hydrolysis process catalyzed by plasmin, a proteolytic enzyme formed from plasminogen. Plasminogen is cleaved by tissue-type (tPA) and urokinase-type (uPA) activators and inhibited by the plasminogen activator inhibitors type-1 (PAI-1) and type-2 (PAI-2). The whole process maintains blood hemostasis. This study aims to assess PAI-1, PAI-2, tPA and uPA mRNA expression in primary cultured human umbilical vein endothelial cells (HUVEC) isolated and cultured from healthy, HD and PRE women. Results show that PAI-1 and PAI-2 mRNA decreased in HD-HUVEC, whereas PAI-1 and uPA decreased in PRE-HUVEC cultures compared to control ones. Notably, the expression ratio between pro- and anti-fibrinolytic actors remained unchanged among the studied groups. It seems that newborn's hemostasis is maintained balanced probably by a compensatory mechanism that involves changes in the fibrinolysis gene expression profile. The real impact of these changes in mRNA expression is unknown, however, it is suggested that these changes could be associated with an increased predisposition to vascular disease development in the progeny. Copyright © 2018. Published by Elsevier Ltd.

The plasminogen activator system in the ovine placentome during late gestation and stage-two of parturition.

PubMed

McNeel, Anthony K; Cushman, Robert A; Vallet, Jeffrey L

2013-06-01

The process of placental separation is not completely understood. In domestic animals, especially cattle, it is important that expulsion of the fetal membranes takes place in a timely manner in order to achieve maximal reproductive efficiency. The activity of the matrix-metalloprotease (MMP) family of proteases is known to be reduced in placentomes from cases of retained placenta. Members of the MMP family are known to be activated by the plasminogen activator (PA) family of proteases. We hypothesized that the expression and activity of the PA family increase in the cotyledon and/or caruncle as parturition approaches, with maximal expression and activity at parturition. To test this hypothesis, we performed reverse-transcriptase quantitative PCR and plasminogen-casein zymography to detect the presence and activity of PA family members in the placentome leading up to and during parturition in spontaneous and dexamethasone-induced parturient ewes. The results from our experiments indicated that serine proteases inhibitor E1 (SERPINE1) mRNA abundance in the cotyledon was different between treatment groups (P = 0.0002). In the caruncle, gene expression for plasminogen activator urokinase-type (PLAU) was different (P = 0.0154), and there was a strong trend for differences in SERPINE1 expression (P = 0.0565). These results demonstrate that expression of the PA system in the placentome changes from late pregnancy to parturition, and the presence or activity of these enzymes may occur after fetal expulsion.

Inhibitory Monoclonal Antibodies against Mouse Proteases Raised in Gene-Deficient Mice Block Proteolytic Functions in vivo

PubMed Central

Lund, Ida K.; Rasch, Morten G.; Ingvarsen, Signe; Pass, Jesper; Madsen, Daniel H.; Engelholm, Lars H.; Behrendt, Niels; Høyer-Hansen, Gunilla

2012-01-01

Identification of targets for cancer therapy requires the understanding of the in vivo roles of proteins, which can be derived from studies using gene-targeted mice. An alternative strategy is the administration of inhibitory monoclonal antibodies (mAbs), causing acute disruption of the target protein function(s). This approach has the advantage of being a model for therapeutic targeting. mAbs for use in mouse models can be obtained through immunization of gene-deficient mice with the autologous protein. Such mAbs react with both species-specific epitopes and epitopes conserved between species. mAbs against proteins involved in extracellular proteolysis, including plasminogen activators urokinase plasminogen activator (uPA), tissue-type plasminogen activator (tPA), their inhibitor PAI-1, the uPA receptor (uPAR), two matrix metalloproteinases (MMP9 and MMP14), as well as the collagen internalization receptor uPARAP, have been developed. The inhibitory mAbs against uPA and uPAR block plasminogen activation and thereby hepatic fibrinolysis in vivo. Wound healing, another plasmin-dependent process, is delayed by an inhibitory mAb against uPA in the adult mouse. Thromboembolism can be inhibited by anti-PAI-1 mAbs in vivo. In conclusion, function-blocking mAbs are well-suited for targeted therapy in mouse models of different diseases, including cancer. PMID:22754528

Diagnostic value of decoy receptor 3 combined with procalcitonin and soluble urokinase-type plasminogen activator receptor for sepsis.

PubMed

Zhao, Jing-Jing; Lou, Xiao-Li; Chen, Hong-Wei; Zhu, Feng-Ting; Hou, Yan-Qiang

2018-01-01

The levels of decoy receptor 3 (DcR3), soluble urokinase type plasminogen activator receptor (suPAR) and procalcitonin (PCT) are significantly increased in sepsis. We investigated the diagnostic value of DcR3 combined with suPAR and PCT in sepsis. Patients with sepsis, non-infectious systemic inflammatory response comprehensive syndrome (SIRS) and healthy controls were recruited according to the diagnostic standard. We measured DcR3, suPAR, PCT, interleukin-6 (IL-6) and C-reactive protein (CRP), and the diagnostic value was evaluated by receiver operating characteristics (ROC) curves. In our analysis, serum DcR3, suPAR and PCT levels of the sepsis group were significantly higher than those of the SIRS and control groups. However, IL-6, CRP and WBC showed no significant difference between the SIRS group and the sepsis group. The serum DcR3 level was positively correlated with the serum suPAR level ( r  = 0.37, p  = 0.0022) and PCT level ( r  = 0.37, p  = 0.0021). Using DcR3, suPAR and PCT to distinguish SIRS from sepsis, the area under the curve (AUC) values were 0.892, 0.778 and 0.692. When DcR3, suPAR and PCT combined were used for diagnosis of sepsis, the AUC was 0.933, at a cut-off point of 0.342. This combination improved the sensitivity and specificity of diagnosis of sepsis, suggesting that use of the combination of three indexes enhanced the efficiency of sepsis diagnosis.

The Association of Plasminogen Activator Inhibitor Type 1 (PAI-1) Level and PAI-1 4G/5G Gene Polymorphism with the Formation and the Grade of Endometrial Cancer.

PubMed

Yıldırım, Malik Ejder; Karakuş, Savas; Kurtulgan, Hande Küçük; Kılıçgün, Hasan; Erşan, Serpil; Bakır, Sevtap

2017-08-01

Plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor (Serpine 1), and it inhibits both tissue plasminogen activator and urokinase plasminogen activator which are important in fibrinolysis. We aimed to find whether there is a possible association between PAI-1 level, PAI-1 4G/5G polymorphism, and endometrial cancer. PAI-1 levels in peripheral blood were determined in 82 patients with endometrial carcinoma and 76 female healthy controls using an enzyme-linked immunoassay (ELISA). Then, the genomic DNA was extracted and screened by reverse hybridization procedure (Strip assay) to detect PAI 1 4G/5G polymorphism. The levels of PAI-1 in the patients were higher statistically in comparison to controls (P < 0.001). The distribution of PAI-1 4G/5G polymorphism was quite different between patients and controls (P = 0.008), and 4G allelic frequency was significantly higher in the patients of endometrial cancer than in controls (P = 0.026). We found significant difference between Grade 1 and Grade 2+3 patients in terms of the PAI-1 levels (P = 0.047). There was no association between PAI-1 4G/5G polymorphism and the grades of endometrial cancer (P = 0.993). Our data suggest that the level of PAI-1 and PAI-1 4G/5G gene polymorphism are effective in the formation of endometrial cancer. PAI-1 levels are also associated with the grades of endometrial cancer.

Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity.

PubMed

Mitchell, Joanne L; Lionikiene, Ausra S; Georgiev, Georgi; Klemmer, Anja; Brain, Chelsea; Kim, Paul Y; Mutch, Nicola J

2016-12-15

Activated factor XII (FXIIa) has plasminogen activator capacity but its relative contribution to fibrinolysis is considered marginal compared with urokinase and tissue plasminogen activator. Polyphosphate (polyP) is released from activated platelets and mediates FXII activation. Here, we investigate the contribution of polyP to the plasminogen activator function of αFXIIa. We show that both polyP 70 , of the chain length found in platelets (60-100 mer), and platelet-derived polyP significantly augment the plasminogen activation capacity of αFXIIa. PolyP 70 stimulated the autoactivation of FXII and subsequent plasminogen activation, indicating that once activated, αFXIIa remains bound to polyP 70 Indeed, complex formation between polyP 70 and αFXIIa provides protection against autodegradation. Plasminogen activation by βFXIIa was minimal and not enhanced by polyP 70 , highlighting the importance of the anion binding site. PolyP 70 did not modulate plasmin activity but stimulated activation of Glu and Lys forms of plasminogen by αFXIIa. Accordingly, polyP 70 was found to bind to FXII, αFXIIa, and plasminogen, but not βFXIIa. Fibrin and polyP 70 acted synergistically to enhance αFXIIa-mediated plasminogen activation. The plasminogen activator activity of the αFXIIa-polyP 70 complex was modulated by C1 inhibitor and histidine-rich glycoprotein, but not plasminogen activator inhibitors 1 and 2. Platelet polyP and FXII were found to colocalize on the activated platelet membrane in a fibrin-dependent manner and decorated fibrin strands extending from platelet aggregates. We show that in the presence of platelet polyP and the downstream substrate fibrin, αFXIIa is a highly efficient and favorable plasminogen activator. Our data are the first to document a profibrinolytic function of platelet polyP. © 2016 by The American Society of Hematology.

Enhanced expression of the urokinase-type plasminogen activator gene and reduced colony formation in soft agar by ectopic expression of PU.1 in HT1080 human fibrosarcoma cells.

PubMed Central

Kondoh, N.; Yamada, T.; Kihara-Negishi, F.; Yamamoto, M.; Oikawa, T.

1998-01-01

To investigate the cell biological function of PU.1, a member of the Ets family of transcription factors, a vector capable of expressing the protein was transfected into HT1080 human fibrosarcoma cells. Exogenous expression of PU.1 in HT1080 cells reduced colony-forming efficiency but stimulated cell migration in soft agar, although it did not affect cell growth in adherent culture. Expression of the urokinase-type plasminogen activator (uPA) mRNA, which is known to be correlated with cell migration and invasion, was enhanced in PU.1 transfectants compared with mock transfectants. Run-on analysis demonstrated that uPA transcription was unaffected by PU.1, suggesting that this enhancement mainly occurs at a post-transcriptional level. On the other hand, treatment of HT1080 cells with the synthetic glucocorticoid dexamethasone (DEX; 10(-7) M) significantly reduced uPA gene expression at a transcriptional level. Furthermore, DEX inhibited cell migration in soft agar without affecting cell growth. These negative effects of DEX on uPA expression and cell migration were alleviated by the expression of PU.1 in HT1080 cells, whereas expression of the N-ras oncogene, which is responsible for maintenance of the transformed phenotypes in HT1080 cells, was unaffected by PU.1 expression or DEX treatment in the cells. Our results suggest that expression of PU.1 can stimulate uPA gene expression at the post-transcriptional level, which may subsequently lead to activation of cell motility and/or reduced cell-cell adhesion, but reduces anchorage-independent growth of HT1080 cells. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9743289

Quebec platelet disorder is linked to the urokinase plasminogen activator gene (PLAU) and increases expression of the linked allele in megakaryocytes

PubMed Central

Diamandis, Maria; Paterson, Andrew D.; Rommens, Johanna M.; Veljkovic, D. Kika; Blavignac, Jessica; Bulman, Dennis E.; Waye, John S.; Derome, Francine; Rivard, Georges E.

2009-01-01

Quebec platelet disorder (QPD) is an autosomal dominant disorder with high penetrance that is associated with increased risks for bleeding. The hallmark of QPD is a gain-of-function defect in fibrinolysis due to increased platelet content of urokinase plasminogen activator (uPA) without systemic fibrinolysis. We hypothesized that increased expression of uPA by differentiating QPD megakaryocytes is linked to PLAU. Genetic marker analyses indicated that QPD was significantly linked to a 2-Mb region on chromosome 10q containing PLAU with a maximum multipoint logarithm of the odds (LOD) score of +11 between markers D10S1432 and D10S1136. Analysis of PLAU by sequencing and Southern blotting excluded mutations within PLAU and its known regulatory elements as the cause of QPD. Analyses of uPA mRNA indicated that QPD distinctly increased transcript levels of the linked PLAU allele with megakaryocyte differentiation. These findings implicate a mutation in an uncharacterized cis element near PLAU as the cause of QPD. PMID:18988861

Quebec platelet disorder is linked to the urokinase plasminogen activator gene (PLAU) and increases expression of the linked allele in megakaryocytes.

PubMed

Diamandis, Maria; Paterson, Andrew D; Rommens, Johanna M; Veljkovic, D Kika; Blavignac, Jessica; Bulman, Dennis E; Waye, John S; Derome, Francine; Rivard, Georges E; Hayward, Catherine P M

2009-02-12

Quebec platelet disorder (QPD) is an autosomal dominant disorder with high penetrance that is associated with increased risks for bleeding. The hallmark of QPD is a gain-of-function defect in fibrinolysis due to increased platelet content of urokinase plasminogen activator (uPA) without systemic fibrinolysis. We hypothesized that increased expression of uPA by differentiating QPD megakaryocytes is linked to PLAU. Genetic marker analyses indicated that QPD was significantly linked to a 2-Mb region on chromosome 10q containing PLAU with a maximum multipoint logarithm of the odds (LOD) score of +11 between markers D10S1432 and D10S1136. Analysis of PLAU by sequencing and Southern blotting excluded mutations within PLAU and its known regulatory elements as the cause of QPD. Analyses of uPA mRNA indicated that QPD distinctly increased transcript levels of the linked PLAU allele with megakaryocyte differentiation. These findings implicate a mutation in an uncharacterized cis element near PLAU as the cause of QPD.

Inhibition of plasminogen activator inhibitor-1 binding to endocytosis receptors of the low-density-lipoprotein receptor family by a peptide isolated from a phage display library

PubMed Central

Jensen, Jan K.; Malmendal, Anders; Schiøtt, Birgit; Skeldal, Sune; Pedersen, Katrine E.; Celik, Leyla; Nielsen, Niels Chr.; Andreasen, Peter A.; Wind, Troels

2006-01-01

The functions of the serpin PAI-1 (plasminogen activator inhibitor-1) are based on molecular interactions with its target proteases uPA and tPA (urokinase-type and tissue-type plasminogen activator respectively), with vitronectin and with endocytosis receptors of the low-density-lipoprotein family. Understanding the significance of these interactions would be facilitated by the ability to block them individually. Using phage display, we have identified the disulfide-constrained peptide motif CFGWC with affinity for natural human PAI-1. The three-dimensional structure of a peptide containing this motif (DVPCFGWCQDA) was determined by liquid-state NMR spectroscopy. A binding site in the so-called flexible joint region of PAI-1 was suggested by molecular modelling and validated through binding studies with various competitors and site-directed mutagenesis of PAI-1. The peptide with an N-terminal biotin inhibited the binding of the uPA–PAI-1 complex to the endocytosis receptors low-density-lipoprotein-receptor-related protein 1A (LRP-1A) and very-low-density-lipoprotein receptor (VLDLR) in vitro and inhibited endocytosis of the uPA–PAI-1 complex in U937 cells. We conclude that the isolated peptide represents a novel approach to pharmacological interference with the functions of PAI-1 based on inhibition of one specific molecular interaction. PMID:16813566

Enhanced venous thrombus resolution in plasminogen activator inhibitor type-2 deficient mice.

PubMed

Siefert, S A; Chabasse, C; Mukhopadhyay, S; Hoofnagle, M H; Strickland, D K; Sarkar, R; Antalis, T M

2014-10-01

The resolution of deep vein thrombosis requires an inflammatory response and mobilization of proteases, such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs), to degrade the thrombus and remodel the injured vein wall. Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor (serpin) with unique immunosuppressive and cell survival properties that was originally identified as an inhibitor of uPA. To investigate the role of PAI-2 in venous thrombus formation and resolution. Venous thrombus resolution was compared in wild-type C57BL/6, PAI-2(-/-) , and PAI-1(-/-) mice using the stasis model of deep vein thrombosis. Formed thrombi were harvested, thrombus weights were recorded, and tissue was analyzed for uPA and MMP activities, PAI-1 expression, and the nature of inflammatory cell infiltration. We found that the absence of PAI-2 enhanced venous thrombus resolution, while thrombus formation was unaffected. Enhanced venous thrombus resolution in PAI-2(-/-) mice was associated with increased uPA activity and reduced levels of PAI-1, with no significant effect on MMP-2 and -9 activities. PAI-1 deficiency resulted in an increase in thrombus resolution similar to PAI-2 deficiency, but additionally reduced venous thrombus formation and altered MMP activity. PAI-2-deficient thrombi had increased levels of the neutrophil chemoattractant CXCL2, which was associated with early enhanced neutrophil recruitment. These data identify PAI-2 as a novel regulator of venous thrombus resolution, which modulates several pathways involving both inflammatory and uPA activity mechanisms, distinct from PAI-1. Further examination of these pathways may lead to potential therapeutic prospects in accelerating thrombus resolution. © 2014 International Society on Thrombosis and Haemostasis.

The Plasminogen Activation System Promotes Dendritic Spine Recovery and Improvement in Neurological Function After an Ischemic Stroke

PubMed Central

Jeanneret, Valerie; Yepes, Manuel

2016-01-01

Advances in neurocritical care and interventional neuroradiology have led to a significant decrease in acute ischemic stroke (AIS) mortality. In contrast, due to the lack of an effective therapeutic strategy to promote neuronal recovery among AIS survivors, cerebral ischemia is still a leading cause of disability in the world. Ischemic stroke has a harmful impact on synaptic structure and function, and plasticity-mediated synaptic recovery is associated with neurological improvement following an AIS. Dendritic spines (DSs) are specialized dendritic protrusions that receive most of the excitatory input in the brain. The deleterious effect of cerebral ischemia on DSs morphology and function has been associated with impaired synaptic transmission and neurological deterioration. However, these changes are reversible if cerebral blood flow is restored on time, and this recovery has been associated with neurological improvement following an AIS. Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are two serine proteases that besides catalyzing the conversion of plasminogen into plasmin in the intravascular and pericellular environment, respectively, are also are efficient inductors of synaptic plasticity. Accordingly, recent evidence indicates that both, tPA and uPA, protect DSs from the metabolic stress associated with the ischemic injury, and promote their morphological and functional recovery during the recovery phase from an AIS. Here we will review data indicating that plasticity-induced changes in DSs and the associated post-synaptic density play a pivotal role in the recovery process from AIS, making special emphasis on the role of tPA and uPA in this process. PMID:26846991

Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

PubMed Central

Vilhardt, Frederik; Nielsen, Morten; Sandvig, Kirsten; van Deurs, Bo

1999-01-01

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy. PMID:9880335

Monoclonal Antibody Testing for Cancer Metastasis

NASA Technical Reports Server (NTRS)

1993-01-01

Malignant cells are characterized by the ability to invade surrounding normal tissues. Tumor invasion is abetted by proteolytic enzymes that have been correlated with recurrent disease and metastasis. These enzymes are involved in a cascade of proteolytic interactions with other enzymes and inhibitors which allow cancer cells to dissolve surrounding extracellular matrix, thereby enabling the cells to rapidly invade adjacent tissues and migrate to metastatic sites distant from the primary tumor. Among these proteases are the plasminogen activators (PA), collagenase IV, faminase, and in some cases cathepsin D, which together mediate key steps in the invasion process of metastasis. Cells which have the selective advantage for invasion and metastasis are those capable of regulating their proteolytic activity and proliferation. Cells in the process of invasion would be probably down-regulated for proliferation, but subsequent to attachment and adhesion at a distant site, would then be in a proliferative mode, up-regulating DNA replication. Urokinase (uPA) can be present in the tissues in several molecular forms. The inactive proenzyme is a single chain protein (scuPA) that is cleaved at Lys. 158 to form the double chain, high molecular weight active form (HMW-uPA) of 54 kD. A low molecular weight form (LMW-uPA) can also be produced by cleavage of the HMW-U PA at Lys. 135 - Lys. 136 giving a 35 kD active enzyme. Recently, it has been shown that the HMW active form of urokinase, bound to the tumor cell membrane, is responsible for the local lysis of the extracellular matrix, hence the tissue invasion mechanism for metastasis (Andreasen et al, 19861. Receptor- (membrane) bound uPA is twice as efficient (catalytically) as free fluid-phase uPA. Tho unbound uPA and the LMW form is not responsible for most of the local dissolution of extracellular matrix in the immediate vicinity of the metastatic tumor cell. High levels of urokinase (greater than 3.49 ng/mg of total protein) extracted from breast tumor tissues have recently been shown, together with plasminogen activator inhibitor 1 (PAI-1), to be a good prognostic indicator for high risk of recurrence and shorter patient survival times. In this project, we have attempted to develop immunocytochemical methodologies for the clinical assessment of the expression of urokinase plasminogen activator, which has been implicated to be important for initial steps in tumor invasion, and to relate it to cell proliferation and DNA replication at the single-cell level.

Bolus dose response characteristics of single chain urokinase plasminogen activator and tissue plasminogen activator in a dog model of arterial thrombosis.

PubMed

Badylak, S F; Voytik, S; Klabunde, R E; Henkin, J; Leski, M

1988-11-15

Tissue plasminogen activator (t-PA) and single chain urokinase-plasminogen activator (scu-PA) are relatively "fibrin-specific" thrombolytic drugs with short plasma half lives of 6-8 minutes. Most treatment regimens with these agents utilize a bolus injection followed by continuous drug infusion, usually combined with anticoagulant therapy. The purpose of this study was to establish the dose-response characteristics for scu-PA and t-PA, when given as a single intravenous bolus injection, in a dog model of arterial thrombosis. Eight groups of 6 dogs each were given one of the following doses of scu-PA (mg/kg): 0.20, 0.50, 1.00, 2.00; or t-PA: 0.05, 0.10, 0.20; or an equivalent amount of saline (control group). All doses were given as a single bolus injection 60 minutes after formation of a totally occlusive femoral artery thrombus. Thrombolysis was measured by monitoring the continuous decrement of 125I activity from a radiolabelled thrombus. Ninety minutes after drug injection, all scu-PA treated dogs showed greater thrombolysis (30%, 45%, 56%, and 67%, respectively) than the control group (15%, p less than 0.01). The 0.10 and 0.20 mg/kg t-PA treated dogs showed greater thrombolysis (35% and 49%, respectively) than the control group (15%, p less than 0.01). Both scu-PA and t-PA caused a partial and dose-dependent decrease in alpha 2-antiplasmin activity but scu-PA caused a greater depletion (72% vs. 18%, respectively, p less than 0.05) at 60 minutes after the highest dose of drug administration. Both drugs showed a longer than expected thrombolytic effect based upon the known half lives. Neither drug caused significant changes in the prothrombin time, activated partial thromboplastin time, thrombin time, hematocrit, platelet count, or fibrin degradation product concentration. Single bolus injections of scu-PA and t-PA produce safe and effective thrombolysis in this dog model of arterial thrombosis.

Fisetin Inhibits Migration and Invasion of Human Cervical Cancer Cells by Down-Regulating Urokinase Plasminogen Activator Expression through Suppressing the p38 MAPK-Dependent NF-κB Signaling Pathway

PubMed Central

Chou, Ruey-Hwang; Hsieh, Shu-Ching; Yu, Yung-Luen; Huang, Min-Hsien; Huang, Yi-Chang; Hsieh, Yi-Hsien

2013-01-01

Fisetin (3,3’,4’,7-tetrahydroxyflavone), a naturally occurring flavonoid, has been reported to inhibit proliferation and induce apoptosis in several cancer types. However, its effect on the anti-metastatic potential of cervical cancer cells remains unclear. In the present study, we found that fisetin inhibits the invasion and migration of cervical cancer cells. The expression and activity of urokinase plasminogen activator (uPA) was significantly suppressed by fisetin in a dose-dependent manner. We also demonstrated that fisetin reduces the phosphorylation of p38 MAPK, but not that of ERK1/2, JNK1/2, or AKT. Addition of a p38 MAPK inhibitor, SB203580, further enhanced the inhibitory effect of fisetin on the expression and activity of uPA and the invasion and motility in cervical cancer cells. Fisetin suppressed the TPA (tetradecanoylphorbol-13-acetate)-induced activation of p38 MAPK and uPA, and inhibited the TPA-enhanced migratory and invasive abilities. Furthermore, the promoter activity of the uPA gene was dramatically repressed by fisetin, which disrupted the nuclear translocation of NF-κB and its binding amount on the promoter of the uPA gene, and these suppressive effects could be further enhanced by SB203580. This study provides strong evidence for the molecular mechanism of fisetin in inhibiting the aggressive phenotypes by repression of uPA via interruption of p38 MAPK-dependent NF-κB signaling pathway in cervical cancer cells and thus contributes insight to the potential of using fisetin as a therapeutic strategy against cervical cancer by inhibiting migration and invasion. PMID:23940799

Cavity Versus Ligand Shape Descriptors: Application to Urokinase Binding Pockets.

PubMed

Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Camproux, Anne-Claude; Petitjean, Michel

2017-11-01

We analyzed 78 binding pockets of the human urokinase plasminogen activator (uPA) catalytic domain extracted from a data set of crystallized uPA-ligand complexes. These binding pockets were computed with an original geometric method that does NOT involve any arbitrary parameter, such as cutoff distances, angles, and so on. We measured the deviation from convexity of each pocket shape with the pocket convexity index (PCI). We defined a new pocket descriptor called distributional sphericity coefficient (DISC), which indicates to which extent the protein atoms of a given pocket lie on the surface of a sphere. The DISC values were computed with the freeware PCI. The pocket descriptors and their high correspondences with ligand descriptors are crucial for polypharmacology prediction. We found that the protein heavy atoms lining the urokinases binding pockets are either located on the surface of their convex hull or lie close to this surface. We also found that the radii of the urokinases binding pockets and the radii of their ligands are highly correlated (r = 0.9).

Predicting Mortality in African Americans With Type 2 Diabetes Mellitus: Soluble Urokinase Plasminogen Activator Receptor, Coronary Artery Calcium, and High-Sensitivity C-Reactive Protein.

PubMed

Hayek, Salim S; Divers, Jasmin; Raad, Mohamad; Xu, Jianzhao; Bowden, Donald W; Tracy, Melissa; Reiser, Jochen; Freedman, Barry I

2018-05-01

Type 2 diabetes mellitus is a major risk factor for cardiovascular disease; however, outcomes in individual patients vary. Soluble urokinase plasminogen activator receptor (suPAR) is a bone marrow-derived signaling molecule associated with adverse cardiovascular and renal outcomes in many populations. We characterized the determinants of suPAR in African Americans with type 2 diabetes mellitus and assessed whether levels were useful for predicting mortality beyond clinical characteristics, coronary artery calcium (CAC), and high-sensitivity C-reactive protein (hs-CRP). We measured plasma suPAR levels in 500 African Americans with type 2 diabetes mellitus enrolled in the African American-Diabetes Heart Study. We used Kaplan-Meier curves and Cox proportional hazards models adjusting for clinical characteristics, CAC, and hs-CRP to examine the association between suPAR and all-cause mortality. Last, we report the change in C-statistics comparing the additive values of suPAR, hs-CRP, and CAC to clinical models for prediction of mortality. The suPAR levels were independently associated with female sex, smoking, insulin use, decreased kidney function, albuminuria, and CAC. After a median 6.8-year follow-up, a total of 68 deaths (13.6%) were recorded. In a model incorporating suPAR, CAC, and hs-CRP, only suPAR was significantly associated with mortality (hazard ratio 2.66, 95% confidence interval 1.63-4.34). Addition of suPAR to a baseline clinical model significantly improved the C-statistic for all-cause death (Δ0.05, 95% confidence interval 0.01-0.10), whereas addition of CAC or hs-CRP did not. In African Americans with type 2 diabetes mellitus, suPAR was strongly associated with mortality and improved risk discrimination metrics beyond traditional risk factors, CAC and hs-CRP. Studies addressing the clinical usefulness of measuring suPAR concentrations are warranted. © 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Plasminogen activator inhibitor links obesity and thrombotic cerebrovascular diseases: The roles of PAI-1 and obesity on stroke.

PubMed

Chen, Rui; Yan, Jinchuan; Liu, Peijing; Wang, Zhongqun; Wang, Cuiping

2017-06-01

One of the global socioeconomic phenomena occurred during the last decades is the increased prevalence of obesity, with direct consequence on the risk of developing thrombotic disorders. As the physiological inhibitor of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) is well known for its role in fibrinolysis. More and more evidences have shown that PAI-1 involves in physiopathologic mechanisms of many diseases and metabolic disorder. Increased serum level of PAI-1 has been observed in obesity and it also contributes to the development of adipose tissue and then has effects on obesity. Meantime, obesity affects also the PAI-1 levels. These evidences indicate the complicated interaction between PAI-1 and obesity. Many clinic studies have confirmed that obesity relates to the stroke outcome although there are many contradictory results. Simultaneously, correlation is found between plasma PAI-1 and thrombotic cerebrovascular diseases. This article reviews contemporary knowledge regarding the complex interplay of obesity, PAI-1 and stroke.

The fibrinolytic mechanism of defibrotide: effect of defibrotide on plasmin activity.

PubMed

Echart, Cinara L; Graziadio, Barbara; Somaini, Simona; Ferro, Laura I; Richardson, Paul G; Fareed, Jawed; Iacobelli, Massimo

2009-12-01

Fibrinolytic activity has been shown to be reduced in many vascular diseases, including hepatic veno-occlusive disease after stem cell transplantation, a microangiopathy characterized by sinusoidal endothelial cell injury. Defibrotide is a polydisperse oligonucleotide with antithrombotic, profibrinolytic, anti-ischemic, and antiadhesive properties. Numerous clinical studies have shown promising activity of defibrotide in the treatment and prevention of veno-occlusive disease, with minimal toxicity. In corollary laboratory studies, defibrotide has been shown to decrease plasminogen activator inhibitor-1, increase tissue plasminogen activator levels, and increase overall plasma fibrinolytic activity in patients. Plasmin, a potent and nonspecific serine protease, plays a pivotal role in fibrinolysis by virtue of its ability to effectively degrade fibrin clots. In this study, defibrotide increases the activity of plasmin in hydrolyzing its substrate in a dose-dependent and length-dependent manner. Similar concentration-dependent effects of defibrotide were observed when plasmin was generated by tissue plasminogen activator or urokinase activation of plasminogen. In contrast, defibrotide had no direct effect on the activation of plasminogen to plasmin. Defibrotide was also able to enhance the activity of plasmin in degrading fibrin clot formed from fibrinogen, plasminogen, and thrombin. This effect was also concentration-dependent and directly correlated with the enzymatic activity of plasmin. This study therefore demonstrates that defibrotide is capable of enhancing the activity of plasmin and so contributes to its fibrinolytic activity. Taken together, these results support the effect of defibrotide in restoring the fibrinolytic vascular phenotype, in microangiopathic conditions such as veno-occlusive disease.

PET imaging of urokinase-type plasminogen activator receptor (uPAR) in prostate cancer: current status and future perspectives.

PubMed

Skovgaard, Dorthe; Persson, Morten; Kjaer, Andreas

2016-01-01

Overexpression of urokinase-type plasminogen activator receptors (uPAR) represents an important biomarker for aggressiveness in most common malignant diseases, including prostate cancer (PC). Accordingly, uPAR expression either assessed directly in malignant PC tissue or assessed directly in plasma (intact/cleaved forms)-provides independent additional clinical information to that contributed by PSA, Gleason score, and other relevant pathological and clinical parameters. In this respect, non-invasive molecular imaging by positron emission tomography (PET) offers a very attractive technology platform, which can provide the required quantitative information on the uPAR expression profile, without the need for invasive procedures and the risk of missing the target due to tumor heterogeneity. These observations support non-invasive PET imaging of uPAR in PC as a clinically relevant diagnostic and prognostic imaging method. In this review, we will focus on the recent development of uPAR PET and the relevance within prostate cancer imaging. Novel antibody and small-molecule radiotracers-targeting uPAR, including a series of uPAR-targeting PET ligands, based on the high affinity peptide ligand AE105, have been synthesized and tested in vitro and in vivo in preclinical murine xenograft models and, recently, in a first-ever clinical uPAR PET study in cancer patients, including patients with PC. In this phase I study, a high and specific uptake of the tracer 64 Cu-DOTA-AE105 was found in both primary tumors and lymph node metastases. The results are encouraging and support large-scale clinical trials to determine the utility of uPAR PET in the management of patients with PC with the goal of improving outcome.

Angiogenesis Research to Improve Therapies for Vascular Leak Syndromes, Intra-abdominal Adhesions, and Arterial Injuries

DTIC Science & Technology

2008-04-01

small-cell lung cancer; TFP1, transferrin pseudogene 1; TGFβ, transforming growth factor-β; TNF, tumour- necrosis factor; uPA, urokinase-type plasminogen...Cell 79, 315–328 (1994). 23. Frater-Schroder, M., Risau, W., Hallmann, R., Gautschi, P. & Bohlen, P. Tumor necrosis factor type α, a potent inhibitor...relatively thin (skin) or avascular (cartilage) tissues, where post- implantation vascularization from the host is sufficient. To overcome the problem

THE ENHANCEMENT OF CHLOROFORM-INDUCED PLASMA PROTEOLYTIC ACTIVITY BY EPSILON AMINOCAPROIC ACID

PubMed Central

Donaldson, Virginia H.; Ratnoff, Oscar D.

1962-01-01

The proteolytic activity in chloroform-treated plasma euglobulins has been attributed to plasmin. Plasmin can digest both casein and fibrin. Epsilon aminocaproic acid, which inhibits the activation of plasminogen, the precursor of plasmin, by streptokinase, urokinase, and tissue activators enhanced the development of casein hydrolytic activity in a mixture of chloroform and plasma euglobulins. Fibrinolytic activity was also enhanced, but this was evident only if the epsilon aminocaproic acid was removed from the chloroform-treated euglobulins prior to assay. The reasons for the paradoxical enhancement of chloroform-induced casein hydrolysis by euglobulins containing epsilon aminocaproic acid are unclear. However, studies of optimal pH, heat stability, and the effect of ionic strength on the activation of the precursor of this proteolytic enzyme do not differentiate it from plasminogen. PMID:13887179

Soluble Urokinase-Type Plasminogen Activator Receptor Improves Risk Prediction in Patients With Chronic Heart Failure.

PubMed

Koller, Lorenz; Stojkovic, Stefan; Richter, Bernhard; Sulzgruber, Patrick; Potolidis, Christos; Liebhart, Florian; Mörtl, Deddo; Berger, Rudolf; Goliasch, Georg; Wojta, Johann; Hülsmann, Martin; Niessner, Alexander

2017-04-01

This study investigated the predictive value of soluble urokinase-type plasminogen activator receptor (suPAR) in patients with chronic heart failure (CHF). SuPAR originates from proteolytic cleavage of the membrane-bound receptor from activated immune and endothelial cells and reflects the level of immune activation. As inflammation plays a crucial role in the complex pathophysiology of CHF, we hypothesized that suPAR might be a suitable prognostic biomarker in patients with CHF. SuPAR levels were determined in 319 patients with CHF admitted to our outpatient department for heart failure and in a second cohort consisting of 346 patients with CHF, for validation. During a median follow-up time of 3.2 years, 119 patients (37.3%) died. SuPAR was a strong predictor of mortality with a crude hazard ratio (HR) per increase of 1 SD (HR per 1 SD) of 1.96 (95% confidence interval [CI]: 1.63 to 2.35; p < 0.001) in univariate analysis and remained significant after comprehensive multivariate adjustment with an adjusted HR per 1 SD of 1.38 (95% CI: 1.04 to 1.83; p = 0.026). SuPAR added prognostic value beyond the multivariate model indicated by improvements in C-statistics (area under the curve: 0.72 vs 0.74, respectively; p = 0.02), the category-free net reclassification index (24.9%; p = 0.032), and the integrated discrimination improvement (0.011; p = 0.05). Validation in the second cohort yielded consistent results. SuPAR is a strong and independent predictor of mortality in patients with CHF, potentially suitable to refine risk assessment in this vulnerable group of patients. Our results emphasize the impact of immune activation on survival in patients with CHF. Copyright © 2017 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Urokinase production by electrophoretically separated cultured human embryonic kidney cells

NASA Technical Reports Server (NTRS)

Kunze, M. E.; Plank, L. D.; Giranda, V.; Sedor, K.; Todd, P. W.

1985-01-01

Urokinase is a plasminogen activator found in urine. Relatively pure preparations have been tested in Europe, Japan and the United States for the treatment of deep vein thrombosis and other dangerous blood clots. Human embryonic kidney cell cultures have been found to produce urokinase at much higher concentrations, but less than 5% of the cells in typical cultures are producers. Since human diploid cells become senescent in culture the selection of clones derived from single cells will not provide enough material to be useful, so a bulk purification method is needed for the isolation of urokinase producing cell populations. Preparative cell electrophoresis was chosen as the method, since evidence exists that human embryonic cell cultures are richly heterogeneous with respect to electrophoretic mobility, and preliminary electrophoretic separations on the Apollo-Soyuz space flight produced cell populations that were rich in urokinase production. Similarly, erythropoietin is useful in the treatment of certain anemias and is a kidney cell duct, and electrophoretically enriched cell populations producing this product have been reported. Thus, there is a clear need for diploid human cells that produce these products, and there is evidence that such cells should be separable by free-flow cell electrophoresis.

Successful silencing of plasminogen activator inhibitor-1 in human vascular endothelial cells using small interfering RNA.

PubMed

Hecke, Anneke; Brooks, Hilary; Meryet-Figuière, Matthieu; Minne, Stephanie; Konstantinides, Stavros; Hasenfuss, Gerd; Lebleu, Bernard; Schäfer, Katrin

2006-05-01

Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI-1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis.

Characterization of a Novel Class of Polyphenolic Inhibitors of Plasminogen Activator Inhibitor-1*

PubMed Central

Cale, Jacqueline M.; Li, Shih-Hon; Warnock, Mark; Su, Enming J.; North, Paul R.; Sanders, Karen L.; Puscau, Maria M.; Emal, Cory D.; Lawrence, Daniel A.

2010-01-01

Plasminogen activator inhibitor type 1, (PAI-1) the primary inhibitor of the tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, has been implicated in a wide range of pathological processes, making it an attractive target for pharmacologic inhibition. Currently available small-molecule inhibitors of PAI-1 bind with relatively low affinity and do not inactivate PAI-1 in the presence of its cofactor, vitronectin. To search for novel PAI-1 inhibitors with improved potencies and new mechanisms of action, we screened a library selected to provide a range of biological activities and structural diversity. Five potential PAI-1 inhibitors were identified, and all were polyphenolic compounds including two related, naturally occurring plant polyphenols that were structurally similar to compounds previously shown to provide cardiovascular benefit in vivo. Unique second generation compounds were synthesized and characterized, and several showed IC50 values for PAI-1 between 10 and 200 nm. This represents an enhanced potency of 10–1000-fold over previously reported PAI-1 inactivators. Inhibition of PAI-1 by these compounds was reversible, and their primary mechanism of action was to block the initial association of PAI-1 with a protease. Consistent with this mechanism and in contrast to previously described PAI-1 inactivators, these compounds inactivate PAI-1 in the presence of vitronectin. Two of the compounds showed efficacy in ex vivo plasma and one blocked PAI-1 activity in vivo in mice. These data describe a novel family of high affinity PAI-1-inactivating compounds with improved characteristics and in vivo efficacy, and suggest that the known cardiovascular benefits of dietary polyphenols may derive in part from their inactivation of PAI-1. PMID:20061381

Urokinase receptor is associated with the components of the JAK1/STAT1 signaling pathway and leads to activation of this pathway upon receptor clustering in the human kidney epithelial tumor cell line TCL-598.

PubMed

Koshelnick, Y; Ehart, M; Hufnagl, P; Heinrich, P C; Binder, B R

1997-11-07

The urokinase-type plasminogen activator (uPA) binds to cells via a specific receptor attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Despite the lack of a transmembrane domain, the urokinase receptor (uPAR) is capable of transducing extracellular signals affecting growth, migration, and adhesion. Several Tyr kinases of the src family as well as beta1, beta2, and beta3 integrins were found to be associated with the uPAR. We found that in the human kidney epithelial line TCL-598, also components of the JAK1/STAT1 signal transduction pathway including gp130, are associated with uPAR as revealed by coimmunoprecipitation and are co-localized in caveolae. Upon clustering of uPA.uPAR complex by a monoclonal antibody, JAK1 associates with uPAR, which in turn leads to STAT1 phosphorylation, dimerization, specific binding to DNA, and gene activation. To prove the dependence of STAT1 activation on the uPAR, TCL-598 cells were treated with sense and antisense uPAR oligonucleotides. In antisense-treated cells in which uPAR expression was reduced to less then one third, activation of STAT1 by the clustering antibody was abolished while STAT1 activation by interferon-gamma was unaffected. Therefore, in this cell line, uPA.uPAR also utilizes the JAK1/STAT1 pathway for signaling, and gp130 might be the transmembrane adapter for this signal transduction pathway.

Tumor therapy with a urokinase plasminogen activator-activated anthrax lethal toxin alone and in combination with paclitaxel

PubMed Central

Wein, Alexander N.; Liu, Shihui; Zhang, Yi; McKenzie, Andrew T.; Leppla, Stephen H.

2013-01-01

PA-U2, an engineered anthrax protective antigen that is activated by urokinase was combined with wild-type lethal factor in the treatment of Colo205 colon adenocarcinoma in vitro and B16-BL6 mouse melanoma in vitro and in vivo. This therapy was also tested in combination with the small molecule paclitaxel, based on prior reports suggesting synergy between ERK1/2 inhibition and chemotherapeutics. Colo205 was sensitive to PA-U2/LF while B16-BL6 was not. For the combination treatment of B16-BL6, paclitaxel showed a dose response in vitro, but cells remained resistant to PA-U2/LF even in the presence of paclitaxel. In vivo, each therapy slowed tumor progression, and an additive effect between the two was observed. Since LF targets tumor vasculature while paclitaxel is an anti-mitotic, it is possible the agents were acting against different cells in the stroma, precluding a synergistic effect. The engineered anthrax toxin PA-U2/LF warrants further development and testing, possibly in combination with an anti-angiogenesis therapy such as sunitinib or sorafinib. PMID:22843210

Adipose-derived mesenchymal stromal cells from aged patients with coronary artery disease keep mesenchymal stromal cell properties but exhibit characteristics of aging and have impaired angiogenic potential.

PubMed

Efimenko, Anastasia; Dzhoyashvili, Nina; Kalinina, Natalia; Kochegura, Tatiana; Akchurin, Renat; Tkachuk, Vsevolod; Parfyonova, Yelena

2014-01-01

Tissue regeneration is impaired in aged individuals. Adipose-derived mesenchymal stromal cells (ADSCs), a promising source for cell therapy, were shown to secrete various angiogenic factors and improve vascularization of ischemic tissues. We analyzed how patient age affected the angiogenic properties of ADSCs. ADSCs were isolated from subcutaneous fat tissue of patients with coronary artery disease (CAD; n = 64, 43-77 years old) and without CAD (n = 31, 2-82 years old). ADSC phenotype characterized by flow cytometry was CD90(+)/CD73(+)/CD105(+)/CD45(-)/CD31(-) for all samples, and these cells were capable of adipogenic and osteogenic differentiation. ADSCs from aged patients had shorter telomeres (quantitative reverse transcription polymerase chain reaction) and a tendency to attenuated telomerase activity. ADSC-conditioned media (ADSC-CM) stimulated capillary-like tube formation by endothelial cells (EA.hy926), and this effect significantly decreased with the age of patients both with and without CAD. Angiogenic factors (vascular endothelial growth factor, placental growth factor, hepatocyte growth factor, angiopoetin-1, and angiogenin) in ADSC-CM measured by enzyme-linked immunosorbent assay significantly decreased with patient age, whereas levels of antiangiogenic factors thrombospondin-1 and endostatin did not. Expression of angiogenic factors in ADSCs did not change with patient age (real-time polymerase chain reaction); however, gene expression of factors related to extracellular proteolysis (urokinase and its receptor, plasminogen activator inhibitor-1) and urokinase-type plasminogen activator receptor surface expression increased in ADSCs from aged patients with CAD. ADSCs from aged patients both with and without CAD acquire aging characteristics, and their angiogenic potential declines because of decreasing proangiogenic factor secretion. This could restrict the effectiveness of autologous cell therapy with ADSCs in aged patients.

Cavity Versus Ligand Shape Descriptors: Application to Urokinase Binding Pockets

PubMed Central

Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Camproux, Anne-Claude

2017-01-01

Abstract We analyzed 78 binding pockets of the human urokinase plasminogen activator (uPA) catalytic domain extracted from a data set of crystallized uPA–ligand complexes. These binding pockets were computed with an original geometric method that does NOT involve any arbitrary parameter, such as cutoff distances, angles, and so on. We measured the deviation from convexity of each pocket shape with the pocket convexity index (PCI). We defined a new pocket descriptor called distributional sphericity coefficient (DISC), which indicates to which extent the protein atoms of a given pocket lie on the surface of a sphere. The DISC values were computed with the freeware PCI. The pocket descriptors and their high correspondences with ligand descriptors are crucial for polypharmacology prediction. We found that the protein heavy atoms lining the urokinases binding pockets are either located on the surface of their convex hull or lie close to this surface. We also found that the radii of the urokinases binding pockets and the radii of their ligands are highly correlated (r = 0.9). PMID:28570103

SERPINE1: A Molecular Switch in the Proliferation-Migration Dichotomy in Wound-“Activated” Keratinocytes

PubMed Central

Simone, Tessa M.; Higgins, Craig E.; Czekay, Ralf-Peter; Law, Brian K.; Higgins, Stephen P.; Archambeault, Jaclyn; Kutz, Stacie M.; Higgins, Paul J.

2014-01-01

Significance: A highly interactive serine protease/plasmin/matrix metalloproteinase axis regulates stromal remodeling in the wound microenvironment. Current findings highlight the importance of stringent controls on protease expression and their topographic activities in cell proliferation, migration, and tissue homeostasis. Targeting elements in this cascading network may lead to novel therapeutic approaches for fibrotic diseases and chronic wounds. Recent Advances: Matrix-active proteases and their inhibitors orchestrate wound site tissue remodeling, cell migration, and proliferation. Indeed, the serine proteases urokinase plasminogen activator and tissue-type plasminogen activator (uPA/tPA) and their major phsyiological inhibitor, plasminogen activator inhibitor-1 (PAI-1; serine protease inhibitor clade E member 1 [SERPINE1]), are upregulated in several cell types during injury repair. Coordinate expression of proteolytic enzymes and their inhibitors in the wound bed provides a mechanism for fine control of focal proteolysis to facilitate matrix restructuring and cell motility in complex environments. Critical Issues: Cosmetic and tissue functional consequences of wound repair anomalies affect the quality of life of millions of patients in the United States alone. The development of novel therapeutics to manage individuals most affected by healing anomalies will likely derive from the identification of critical, translationally accessible, control elements in the wound site microenvironment. Future Directions: Activation of the PAI-1 gene early after wounding, its prominence in the repair transcriptome and varied functions suggest a key role in the global cutaneous injury response program. Targeting PAI-1 gene expression and/or PAI-1 function with molecular genetic constructs, neutralizing antibodies or small molecule inhibitors may provide a novel, therapeutically relevant approach, to manage the pathophysiology of wound healing disorders associated with deficient or excessive PAI-1 levels. PMID:24669362

A Small Molecule Inhibitor of Plasminogen Activator Inhibitor-1 Reduces Brain Amyloid-β Load and Improves Memory in an Animal Model of Alzheimer's Disease.

PubMed

Akhter, Hasina; Huang, Wen-Tan; van Groen, Thomas; Kuo, Hui-Chien; Miyata, Toshio; Liu, Rui-Ming

2018-01-01

Alzheimer's disease (AD) is a major cause of dementia in the elderly with no effective treatment. Accumulation of amyloid-β peptide (Aβ) in the brain is a pathological hallmark of AD and is believed to be a central disease-causing and disease-promoting event. In a previous study, we showed that deletion of plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of tissue type and urokinase type plasminogen activators (tPA and uPA), significantly reduced brain Aβ load in APP/PS1 mice, an animal model of familial AD. In this study, we further show that oral administration of TM5275, a small molecule inhibitor of PAI-1, for a period of 6 weeks, inhibits the activity of PAI-1 and increases the activities of tPA and uPA as well as plasmin, which is associated with a reduction of Aβ load in the hippocampus and cortex and improvement of learning/memory function in APP/PS1 mice. Protein abundance of low density lipoprotein related protein-1 (LRP-1), a multi ligand endocytotic receptor involved in transporting Aβ out of the brain, as well as plasma Aβ42 are increased, whereas the expression and processing of full-length amyloid-β protein precursor is not affected by TM5275 treatment in APP/PS1 mice. In vitro studies further show that PAI-1 increases, whereas TM5275 reduces, Aβ40 level in the culture medium of SHSY5Y-APP neuroblastoma cells. Collectively, our data suggest that TM5275 improves memory function of APP/PS1 mice, probably by reducing brain Aβ accumulation through increasing plasmin-mediated degradation and LRP-1-mediated efflux of Aβ in the brain.

Pulmonary Embolism in 2017: How We Got Here and Where Are We Going?

PubMed

Merli, Geno J

2017-09-01

In the 1970s, both the Urokinase Pulmonary Embolism and Urokinase-Streptokinase Pulmonary Embolism trials began the quest to develop thrombolytic therapy for the treatment of acute massive and submassive pulmonary embolism (PE). The goals of these studies were the immediate reduction in clot burden, restoration of hemodynamic stability, and improved survival. Major bleeding became the major barrier for clinicians to employ these therapies. From 1980s to the present time, a number of studies using recombinant tissue-type plasminogen activator for achieving these same above outcomes were completed but major bleeding continued to remain an adoption barrier. Finally, the concept of bringing the thrombolytic agent into the clot has entered the quest for the Holy Grail in the treatment of PE. This article will review all the major trials using peripheral thrombolysis and provide insight into the need for a team approach to pulmonary care (Pulmonary Embolism Response Team), standardization of pulmonary classification, and the need for trials designed for both short- and long-term outcomes using thrombolysis for selected PE populations. Copyright © 2017. Published by Elsevier Inc.

Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies

PubMed Central

Caiolfa, Valeria R.; Zamai, Moreno; Malengo, Gabriele; Andolfo, Annapaola; Madsen, Chris D.; Sutin, Jason; Digman, Michelle A.; Gratton, Enrico; Blasi, Francesco; Sidenius, Nicolai

2007-01-01

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents. PMID:18056417

Discovery of novel urokinase plasminogen activator (uPA) inhibitors using ligand-based modeling and virtual screening followed by in vitro analysis.

PubMed

Al-Sha'er, Mahmoud A; Khanfar, Mohammad A; Taha, Mutasem O

2014-01-01

Urokinase plasminogen activator (uPA)-a serine protease-is thought to play a central role in tumor metastasis and angiogenesis and, therefore, inhibition of this enzyme could be beneficial in treating cancer. Toward this end, we explored the pharmacophoric space of 202 uPA inhibitors using seven diverse sets of inhibitors to identify high-quality pharmacophores. Subsequently, we employed genetic algorithm-based quantitative structure-activity relationship (QSAR) analysis as a competition arena to select the best possible combination of pharmacophoric models and physicochemical descriptors that can explain bioactivity variation within the training inhibitors (r (2) 162 = 0.74, F-statistic = 64.30, r (2) LOO = 0.71, r (2) PRESS against 40 test inhibitors = 0.79). Three orthogonal pharmacophores emerged in the QSAR equation suggesting the existence of at least three binding modes accessible to ligands within the uPA binding pocket. This conclusion was supported by receiver operating characteristic (ROC) curve analyses of the QSAR-selected pharmacophores. Moreover, the three pharmacophores were comparable with binding interactions seen in crystallographic structures of bound ligands within the uPA binding pocket. We employed the resulting pharmacophoric models and associated QSAR equation to screen the national cancer institute (NCI) list of compounds. The captured hits were tested in vitro. Overall, our modeling workflow identified new low micromolar anti-uPA hits.

Serine proteases, inhibitors and receptors in renal fibrosis

PubMed Central

Eddy, Allison A.

2011-01-01

Summary Chronic kidney disease (CKD) is estimated to affect one in eight adults. Their kidney function progressively deteriorates as inflammatory and fibrotic processes damage nephrons. New therapies to prevent renal functional decline must build on basic research studies that identify critical cellular and molecular mediators. Plasminogen activator inhibitor-1 (PAI-1), a potent fibrosis-promoting glycoprotein, is one promising candidate. Absent from normal kidneys, PAI-1 is frequently expressed in injured kidneys. Studies in genetically engineered mice have demonstrated its potency as a pro-fibrotic molecule. Somewhat surprising, its ability to inhibit serine protease activity does not appear to be its primary pro-fibrotic effect in CKD. Both tissue-type plasminogen activator and plasminogen deficiency significantly reduced renal fibrosis severity after ureteral obstruction, while genetic urokinase (uPA) deficiency had no effect. PAI-1 expression is associated with enhanced recruitment of key cellular effectors of renal fibrosis – interstitial macrophages and myofibroblasts. The ability of PAI-1 to promote cell migration involves interactions with the low-density lipoprotein receptor-associate protein-1 and also complex interactions with uPA bound to its receptor (uPAR) and several leukocyte and matrix integrins that associate with uPAR as co-receptors. uPAR is expressed by several cell types in damaged kidneys, and studies in uPAR-deficient mice have shown that its serves a protective role. uPAR mediates additional anti-fibrotic effects - it interacts with specific co-receptors to degrade PAI-1 and extracellular collagens, and soluble uPAR has leukocyte chemoattractant properties. Molecular pathways activated by serine proteases and their inhibitor, PAI-1, are promising targets for future anti-fibrotic therapeutic agents. PMID:19350108

Flow cytometry of human embryonic kidney cells: A light scattering approach

NASA Technical Reports Server (NTRS)

Kunze, M. E.; Goolsby, C. L.; Todd, P. W.; Morrison, D. R.; Lewis, M. L.

1985-01-01

The mammalian kidney contains cells that transport water, convert vitamin D to active forms, synthesize hormones such a renin and erythropoietin, and produce enzymes such as urokinase, a plasminogen activator. Several of these functions are maintained by human embryonic kidney cells (HEK) cultivated in vitro. Biochemical study of these functions in their individual cell types in vitro requires purified populations of cells. Light-scattering activated cell sorting (LACS) was explored as a means of achieving such purifications. It was found that HEK cells at the first 1 to 5 passages in culture were heterogeneous with respect to 2-parameter light scattering intensity distribution, in which combined measurements included forward angle scattering (2.5 to 19 deg), 90 deg scattering, and time-of-flight size measurements. Size was measured at a resolution of 0.15 microns/channel in 256 channels using pulse-height independent pulse-width measurements. Two-parameter distributions combining these measurements were obtained for HEK cell subpopulations that had been purified by microgravity electrophoresis and subsequently propagated in culture. These distributions contained at least 3 subpopulations in all purified fractions, and results of experiments with prepurified cultured HEK cells indicated that subpopulations of living cells that were high in plasminogen-activator activity also contained the highest per cent of cells with high 90 deg light scatter intensity.

A case control study on the structural equation model of the mechanism of coagulation and fibrinolysis imbalance in chronic schistosomiasis.

PubMed

Le, Aiping; Zhang, Lunli; Liu, Wei; Li, Xiaopeng; Ren, Jianwei; Ning, An

2017-02-01

A structural equation model was used for verification with chronic schistosomiasis to investigate the coagulation-anticoagulation system imbalance and to deduce the mechanism of D-dimer (D-D) level elevation in patients with advanced schistosome hepatic disease. We detected the plasma levels of tissue-type fiber plasminogen activator (tPA), urokinase type plasminogen activator (uPA), plasmin-antiplasmin complex (PAP), plasminogen (PLG), antithrombin (AT), plasminogen activator inhibitor 1 (PAI1), D-D, factor VIII: C (FVIII:C), antithrombin-III (AT-III), PLG, protein S (PS), and protein C (PC) in the healthy people as control (69), patients with chronic schistosomiasis (150) or advanced chronic schistosomiasis (90). FVIII, PAP, D-D, tPA, and uPA plasma levels were significantly higher in the chronic group than in the control group and were also significantly higher in the advanced group. However, AT-III, PC, PS, AT, PLG, and PAI1 plasma levels in the advanced and chronic groups were significantly lower than those in the control group. With progression of disease in patients with schistosomiasis japonica, a hypercoagulable state is induced by the coagulation-anticoagulation imbalance, eventually leading to patients with high levels of D-D. Furthermore, we established a structural equation model path of a "chronic schistosomiasis disease stage-(coagulation-anticoagulation-fibrinolysis)-D-D." By using analysis of moment structures (AMOS), it was shown that the chronic schistosomiasis stage was positively related to factor VIII and had negative correlation with AT-III; a good positive correlation with PAP, tPA, and uPA; and a good negative correlation with PLG and PAI1. In addition, our results show that the path coefficient of anticoagulation-fibrinolysis system to the chronic stage of schistosomiasis or D-D levels was significantly higher than that of the coagulation system. In conclusion, the coagulation and fibrinolysis imbalance in patients with chronic schistosomiasis, especially with advanced schistosomiasis, is due to the progression of disease stages.

Temporary dietary iron restriction affects the process of thrombus resolution in a rat model of deep vein thrombosis.

PubMed

Oboshi, Makiko; Naito, Yoshiro; Sawada, Hisashi; Hirotani, Shinichi; Iwasaku, Toshihiro; Okuhara, Yoshitaka; Morisawa, Daisuke; Eguchi, Akiyo; Nishimura, Koichi; Fujii, Kenichi; Mano, Toshiaki; Ishihara, Masaharu; Masuyama, Tohru

2015-01-01

Deep vein thrombosis (DVT) is a major cause of pulmonary thromboembolism and sudden death. Thus, it is important to consider the pathophysiology of DVT. Recently, iron has been reported to be associated with thrombotic diseases. Hence, in this study, we investigate the effects of dietary iron restriction on the process of thrombus resolution in a rat model of DVT. We induced DVT in 8-week-old male Sprague-Dawley rats by performing ligations of their inferior venae cavae. The rats were then given either a normal diet (DVT group) or an iron-restricted diet (DVT+IR group). Thrombosed inferior venae cavae were harvested at 5 days after ligation. The iron-restricted diet reduced venous thrombus size compared to the normal diet. Intrathrombotic collagen content was diminished in the DVT+IR group compared to the DVT group. In addition, intrathrombotic gene expression and the activity of matrix metalloproteinase-9 were increased in the DVT+IR group compared to the DVT group. Furthermore, the DVT+IR group had greater intrathrombotic neovascularization as well as higher gene expression levels of urokinase-type plasminogen activator and tissue-type plasminogen activator than the DVT group. The iron-restricted diet decreased intrathrombotic superoxide production compared to the normal diet. These results suggest that dietary iron restriction affects the process of thrombus resolution in DVT.

Temporary Dietary Iron Restriction Affects the Process of Thrombus Resolution in a Rat Model of Deep Vein Thrombosis

PubMed Central

Oboshi, Makiko; Naito, Yoshiro; Sawada, Hisashi; Hirotani, Shinichi; Iwasaku, Toshihiro; Okuhara, Yoshitaka; Morisawa, Daisuke; Eguchi, Akiyo; Nishimura, Koichi; Fujii, Kenichi; Mano, Toshiaki; Ishihara, Masaharu; Masuyama, Tohru

2015-01-01

Background Deep vein thrombosis (DVT) is a major cause of pulmonary thromboembolism and sudden death. Thus, it is important to consider the pathophysiology of DVT. Recently, iron has been reported to be associated with thrombotic diseases. Hence, in this study, we investigate the effects of dietary iron restriction on the process of thrombus resolution in a rat model of DVT. Methods We induced DVT in 8-week-old male Sprague-Dawley rats by performing ligations of their inferior venae cavae. The rats were then given either a normal diet (DVT group) or an iron-restricted diet (DVT+IR group). Thrombosed inferior venae cavae were harvested at 5 days after ligation. Results The iron-restricted diet reduced venous thrombus size compared to the normal diet. Intrathrombotic collagen content was diminished in the DVT+IR group compared to the DVT group. In addition, intrathrombotic gene expression and the activity of matrix metalloproteinase-9 were increased in the DVT+IR group compared to the DVT group. Furthermore, the DVT+IR group had greater intrathrombotic neovascularization as well as higher gene expression levels of urokinase-type plasminogen activator and tissue-type plasminogen activator than the DVT group. The iron-restricted diet decreased intrathrombotic superoxide production compared to the normal diet. Conclusions These results suggest that dietary iron restriction affects the process of thrombus resolution in DVT. PMID:25962140

Targeting of peptide conjugated magnetic nanoparticles to urokinase plasminogen activator receptor (uPAR) expressing cells

NASA Astrophysics Data System (ADS)

Hansen, Line; Unmack Larsen, Esben Kjær; Nielsen, Erik Holm; Iversen, Frank; Liu, Zhuo; Thomsen, Karen; Pedersen, Michael; Skrydstrup, Troels; Nielsen, Niels Chr.; Ploug, Michael; Kjems, Jørgen

2013-08-01

Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific targeting peptide onto polyethylene glycol (PEG) coated USPIO nanoparticles by click chemistry resulted in a five times higher uptake in vitro in a uPAR positive cell line compared to nanoparticles carrying a non-binding control peptide. In accordance with specific receptor-mediated recognition, a low uptake was observed in the presence of an excess of ATF, a natural ligand for uPAR. The uPAR specific magnetic nanoparticles can potentially provide a useful supplement for tumor patient management when combined with MRI and drug delivery.Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific targeting peptide onto polyethylene glycol (PEG) coated USPIO nanoparticles by click chemistry resulted in a five times higher uptake in vitro in a uPAR positive cell line compared to nanoparticles carrying a non-binding control peptide. In accordance with specific receptor-mediated recognition, a low uptake was observed in the presence of an excess of ATF, a natural ligand for uPAR. The uPAR specific magnetic nanoparticles can potentially provide a useful supplement for tumor patient management when combined with MRI and drug delivery. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr32922d

PAI-1 (Plasminogen Activator Inhibitor-1) Expression Renders Alternatively Activated Human Macrophages Proteolytically Quiescent

PubMed Central

Hohensinner, Philipp J.; Baumgartner, Johanna; Kral-Pointner, Julia B.; Uhrin, Pavel; Ebenbauer, Benjamin; Thaler, Barbara; Doberer, Konstantin; Stojkovic, Stefan; Demyanets, Svitlana; Fischer, Michael B.; Huber, Kurt; Schabbauer, Gernot; Speidl, Walter S.

2017-01-01

Objective— Macrophages are versatile immune cells capable of polarizing into functional subsets depending on environmental stimulation. In atherosclerotic lesions, proinflammatory polarized macrophages are associated with symptomatic plaques, whereas Th2 (T-helper cell type 2) cytokine–polarized macrophages are inversely related with disease progression. To establish a functional cause for these observations, we analyzed extracellular matrix degradation phenotypes in polarized macrophages. Approach and Results— We provide evidence that proinflammatory polarized macrophages rely on membrane-bound proteases including MMP-14 (matrix metalloproteinase-14) and the serine protease uPA (urokinase plasminogen activator) together with its receptor uPAR for extracellular matrix degradation. In contrast, Th2 cytokine alternatively primed macrophages do not show different proteolytic activity in comparison to unpolarized macrophages and lack increased localization of MMP-14 and uPA receptor to the cell membrane. Nonetheless, they express the highest amount of the serine protease uPA. However, uPA activity is blocked by similarly increased expression of its inhibitor PAI-1 (plasminogen activator inhibitor 1). When inhibiting PAI-1 or when analyzing macrophages deficient in PAI-1, Th2 cytokine–polarized macrophages display the same matrix degradation capability as proinflammatory-primed macrophages. Within atherosclerotic lesions, macrophages positive for the alternative activation marker CD206 express high levels of PAI-1. In addition, to test changed tissue remodeling capacities of alternatively activated macrophages, we used a bleomycin lung injury model in mice reconstituted with PAI-1−/− bone marrow. These results supported an enhanced remodeling phenotype displayed by increased fibrosis and elevated MMP activity in the lung after PAI-1 loss. Conclusions— We were able to demonstrate matrix degradation dependent on membrane-bound proteases in proinflammatory stimulated macrophages and a forced proteolytical quiescence in alternatively polarized macrophages by the expression of PAI-1. PMID:28818858

The urokinase receptor-derived cyclic peptide [SRSRY] suppresses neovascularization and intravasation of osteosarcoma and chondrosarcoma cells.

PubMed

Ingangi, Vincenzo; Bifulco, Katia; Yousif, Ali Munaim; Ragone, Concetta; Motti, Maria Letizia; Rea, Domenica; Minopoli, Michele; Botti, Giovanni; Scognamiglio, Giuseppe; Fazioli, Flavio; Gallo, Michele; De Chiara, Annarosaria; Arra, Claudio; Grieco, Paolo; Carriero, Maria Vincenza

2016-08-23

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88-92 is the minimal sequence required to induce cell motility and angiogenesis by interacting with the formyl peptide receptor type 1 (FPR1). In this study, we present evidence that the cyclization of the uPAR88-92 sequence generates a new potent inhibitor of migration, and extracellular matrix invasion of human osteosarcoma and chondrosarcoma cells expressing comparable levels of FPR1 on cell surface. In vitro, the cyclized peptide [SRSRY] prevents formation of capillary-like tubes by endothelial cells co-cultured with chondrosarcoma cells and trans-endothelial migration of osteosarcoma and chondrosarcoma cells. When chondrosarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density and circulating tumor cells in blood samples collected before the sacrifice, were significantly reduced in animals treated daily with i.p-administration of 6 mg/Kg [SRSRY] as compared to animals treated with vehicle only. Our findings indicate that [SRSRY] prevents three key events occurring during the metastatic process of osteosarcoma and chondrosarcoma cells: the extracellular matrix invasion, the formation of a capillary network and the entry into bloodstream.

The urokinase receptor-derived cyclic peptide [SRSRY] suppresses neovascularization and intravasation of osteosarcoma and chondrosarcoma cells

PubMed Central

Ingangi, Vincenzo; Bifulco, Katia; Yousif, Ali Munaim; Ragone, Concetta; Motti, Maria Letizia; Rea, Domenica; Minopoli, Michele; Botti, Giovanni; Scognamiglio, Giuseppe; Fazioli, Flavio; Gallo, Michele; De Chiara, Annarosaria; Arra, Claudio; Grieco, Paolo; Carriero, Maria Vincenza

2016-01-01

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR88–92 is the minimal sequence required to induce cell motility and angiogenesis by interacting with the formyl peptide receptor type 1 (FPR1). In this study, we present evidence that the cyclization of the uPAR88–92 sequence generates a new potent inhibitor of migration, and extracellular matrix invasion of human osteosarcoma and chondrosarcoma cells expressing comparable levels of FPR1 on cell surface. In vitro, the cyclized peptide [SRSRY] prevents formation of capillary-like tubes by endothelial cells co-cultured with chondrosarcoma cells and trans-endothelial migration of osteosarcoma and chondrosarcoma cells. When chondrosarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density and circulating tumor cells in blood samples collected before the sacrifice, were significantly reduced in animals treated daily with i.p-administration of 6 mg/Kg [SRSRY] as compared to animals treated with vehicle only. Our findings indicate that [SRSRY] prevents three key events occurring during the metastatic process of osteosarcoma and chondrosarcoma cells: the extracellular matrix invasion, the formation of a capillary network and the entry into bloodstream. PMID:27323409

RNA Regulation of Estrogen

DTIC Science & Technology

2010-08-01

microtubule-associated protein, RP/EB family, member 3 6.06 211668_s_at PLAU plasminogen activator, urokinase 6.06 207403_at IRS4 insulin receptor...polypeptide 3.48 202410_x_at INS-IGF2 insulin -like growth factor 2 (somatomedin A); insulin ; INS-IGF2 readthrough transcript 3.48 202410_x_at INS insulin -like...growth factor 2 (somatomedin A); insulin ; INS-IGF2 readthrough transcript 3.48 202410_x_at IGF2 insulin -like growth factor 2 (somatomedin A); insulin

Association of tagSNPs in the urokinase-plasminogen activator (PLAU) gene with Alzheimer's disease and associated quantitative traits.

PubMed

Ozturk, Ayla; Minster, Ryan L; DeKosky, Steven T; Kamboh, M Ilyas

2007-01-05

The gene coding for urokinase-plasminogen activator (PLAU) is a strong biological and positional candidate gene for Alzheimer's disease (AD). Previously some studies have examined the role of common variation in the PLAU gene with AD risk but the results have been inconsistent and this inconsistency could have been due to the use of relatively small sample sizes. In this study we evaluated the distribution of four tagSNPs (rs2227562 in intron 5, rs2227564 in exon 6, rs2227571 in intron 9, and rs4065 in 3'UTR) in the PLAU gene in a large case-control study consisting of up to 1,000 AD patients and 697 white control subjects. We examined the role of these tagSNPs with AD risk and quantitative traits of AD, including age-at-onset (AAO), disease duration, and mini-mental state examination (MMSE) scores. The 3'UTR SNP revealed modest significant association with risk (OR = 0.71, 95% CI: 0.53-0.95; P = 0.02), AAO (P = 0.036) and disease duration (P = 0.04) of AD. In addition, the intron 9 SNP also revealed a significant association with AAO (P = 0.01) and disease duration (P = 0.006). Our data on a large number of AD cases and controls suggest that genetic variation in PLAU may affect the risk and AAO of AD.

Receptor-Targeted Nanoparticles for In Vivo Imaging of Breast Cancer

PubMed Central

Yang, Lily; Peng, Xiang-Hong; Wang, Y. Andrew; Wang, Xiaoxia; Cao, Zehong; Ni, Chunchun; Karna, Prasanthi; Zhang, Xinjian; Wood, William C.; Gao, Xiaohu; Nie, Shuming; Mao, Hui

2009-01-01

Purpose Cell surface receptor-targeted magnetic iron oxide (IO) nanoparticles provide molecular magnetic resonance imaging (MRI) contrast agents for improving specificity of the detection of human cancer. Experimental design The present study reports the development of a novel targeted IO nanoparticle using a recombinant peptide containing the amino-terminal fragment (ATF) of urokinase plasminogen activator conjugated to IO nanoparticles (ATF-IO). This nanoparticle targets urokinase plasminogen activator receptor (uPAR), which is overexpressed in breast cancer tissues. Results ATF-IO nanoparticles are able to specifically bind to and be internalized by uPAR-expressing tumor cells. Systemic delivery of ATF-IO nanoparticles into mice bearing subcutaneous and intraperitoneal mammary tumors leads to the accumulation of the particles in tumors, generating a strong MRI contrast detectable by a clinical MRI scanner at a field strength of 3 Tesla. Target specificity of ATF-IO nanoparticles demonstrated by in vivo MRI is further confirmed by near infrared (NIR) fluorescence imaging of the mammary tumors using NIR dye-labeled ATF peptides conjugated to IO nanoparticles. Furthermore, mice administered ATF-IO nanoparticles exhibit lower uptake of the particles in the liver and spleen compared to those receiving non-targeted IO nanoparticles. Conclusions Our results suggest that uPAR-targeted ATF-IO nanoparticles have potential as molecularly-targeted, dual modality imaging agents for in vivo imaging of breast cancer. PMID:19584158

Gastric Expression of Plasminogen Activator Inhibitor (PAI)-1 Is Associated with Hyperphagia and Obesity in Mice

PubMed Central

Kenny, Susan; Gamble, Joanne; Lyons, Suzanne; Vlatković, Nikolina; Dimaline, Rod; Varro, Andrea

2013-01-01

The adipokine plasminogen activator inhibitor (PAI)-1 is increased in plasma of obese individuals and exhibits increased expression in the stomachs of individuals infected with Helicobacter. To investigate the relevance of gastric PAI-1, we used 1.1 kb of the H+/K+β subunit promoter to overexpress PAI-1 specifically in mouse gastric parietal cells (PAI-1-H/Kβ mice). We studied the physiological, biochemical, and behavioral characteristics of these and mice null for PAI-1 or a putative receptor, urokinase plasminogen activator receptor (uPAR). PAI-1-H/Kβ mice had increased plasma concentrations of PAI-1 and increased body mass, adiposity, and hyperphagia compared with wild-type mice. In the latter, food intake was inhibited by cholecystokinin (CCK)8s, but PAI-1-H/Kβ mice were insensitive to the satiating effects of CCK8s. PAI-1-H/Kβ mice also had significantly reduced expression of c-fos in the nucleus tractus solitarius in response to CCK8s and refeeding compared with wild-type mice. Exogenous PAI-1 reversed the effects of CCK8s on food intake and c-fos levels in the nucleus tractus solitarius of wild-type mice, but not uPAR-null mice. Infection of C57BL/6 mice with Helicobacter felis increased gastric abundance of PAI-1 and reduced the satiating effects of CCK8s, whereas the response to CCK8s was maintained in infected PAI-1–null mice. In cultured vagal afferent neurons, PAI-1 inhibited stimulation of neuropeptide Y type 2 receptor (Y2R) expression by CCK8s. Thus, gastric expression of PAI-1 is associated with hyperphagia, moderate obesity, and resistance to the satiating effects of CCK indicating a new role in suppressing signals from the upper gut that inhibit food intake. PMID:23254194

Gastric expression of plasminogen activator inhibitor (PAI)-1 is associated with hyperphagia and obesity in mice.

PubMed

Kenny, Susan; Gamble, Joanne; Lyons, Suzanne; Vlatkovic, Nikolina; Dimaline, Rod; Varro, Andrea; Dockray, Graham J

2013-02-01

The adipokine plasminogen activator inhibitor (PAI)-1 is increased in plasma of obese individuals and exhibits increased expression in the stomachs of individuals infected with Helicobacter. To investigate the relevance of gastric PAI-1, we used 1.1 kb of the H(+)/K(+)β subunit promoter to overexpress PAI-1 specifically in mouse gastric parietal cells (PAI-1-H/Kβ mice). We studied the physiological, biochemical, and behavioral characteristics of these and mice null for PAI-1 or a putative receptor, urokinase plasminogen activator receptor (uPAR). PAI-1-H/Kβ mice had increased plasma concentrations of PAI-1 and increased body mass, adiposity, and hyperphagia compared with wild-type mice. In the latter, food intake was inhibited by cholecystokinin (CCK)8s, but PAI-1-H/Kβ mice were insensitive to the satiating effects of CCK8s. PAI-1-H/Kβ mice also had significantly reduced expression of c-fos in the nucleus tractus solitarius in response to CCK8s and refeeding compared with wild-type mice. Exogenous PAI-1 reversed the effects of CCK8s on food intake and c-fos levels in the nucleus tractus solitarius of wild-type mice, but not uPAR-null mice. Infection of C57BL/6 mice with Helicobacter felis increased gastric abundance of PAI-1 and reduced the satiating effects of CCK8s, whereas the response to CCK8s was maintained in infected PAI-1-null mice. In cultured vagal afferent neurons, PAI-1 inhibited stimulation of neuropeptide Y type 2 receptor (Y2R) expression by CCK8s. Thus, gastric expression of PAI-1 is associated with hyperphagia, moderate obesity, and resistance to the satiating effects of CCK indicating a new role in suppressing signals from the upper gut that inhibit food intake.

Effect of conformational mobility and hydrogen-bonding interactions on the selectivity of some guanidinoaryl-substituted mechanism-based inhibitors of trypsin-like serine proteases.

PubMed

Rai, R; Katzenellenbogen, J A

1992-11-13

Previously, we have reported that some guanidino-substituted alpha- and beta-aryl enol lactones I and II behaved as selective, mechanism-based inhibitors of some trypsin-like proteases (Rai, R.; Katzenellenbogen, J.A. J. Med. Chem., submitted). In this study, we describe the synthesis and kinetic evaluation of some related, guanidino-substituted enol lactones having greater conformational mobility and affording additional hydrogen-bonding sites at the active site. The alpha-aryl-substituted lactones 1 and 2, which have greater conformational mobility in the guanidinoaryl linkage than I, selectively inhibited the trypsin-like enzymes, and they were relatively poor inactivators of alpha-chymotrypsin and human neutrophil elastase (HNE). The iodo enol lactone 2 permanently inactivated trypsin, urokinase, tissue plasminogen activator, and plasmin, showing exceptionally high specificity in its interaction with trypsin and urokinase. The selectivity pattern exhibited by the closely related, conformationally less mobile alpha-aryl-substituted iodo lactone Ib, which was previously shown to be a selective suicide substrate of urokinase and plasmin, provides an interesting comparison. The alpha-benzamido-substituted lactones 3 and 4, which afford an additional site for active-site hydrogen bonding, were found to be very potent alternate substrate inhibitors of trypsin and urokinase. In addition, the iodo lactone 4 permanently inactivated alpha-chymotrypsin. The importance of secondary interactions in increasing the specificities in the case of alpha-chymotrypsin is discussed.

Fibrinolytic and procoagulant activities of Yersinia pestis and Salmonella enterica.

PubMed

Korhonen, T K

2015-06-01

Pla of the plague bacterium Yersinia pestis and PgtE of the enteropathogen Salmonella enterica are surface-exposed, transmembrane β-barrel proteases of the omptin family that exhibit a complex array of interactions with the hemostatic systems in vitro, and both proteases are established virulence factors. Pla favors fibrinolysis by direct activation of plasminogen, inactivation of the serpins plasminogen activator inhibitor-1 and α2-antiplasmin, inactivation of the thrombin-activable fibrinolysis inhibitor, and activation of single-chain urokinase. PgtE is structurally very similar but exhibits partially different functions and differ in expression control. PgtE proteolysis targets control aspects of fibrinolysis, and mimicry of matrix metalloproteinases enhances cell migration that should favor the intracellular spread of the bacterium. Enzymatic activity of both proteases is strongly influenced by the environment-induced variations in lipopolysaccharide that binds to the β-barrel. Both proteases cleave the tissue factor pathway inhibitor and thus also express procoagulant activity. © 2015 International Society on Thrombosis and Haemostasis.

Decoy Plasminogen Receptor Containing a Selective Kunitz-Inhibitory Domain

PubMed Central

2015-01-01

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2′ residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 μM) but not to KD1L17R-VT. Furthermore, KD1L17R-KT inhibited tPA-induced plasma clot fibrinolysis more efficiently than KD1L17R-VT. Additionally, compared to ε-aminocaproic acid KD1L17R-KT was more effective in reducing blood loss in a mouse liver-laceration injury model, where the fibrinolytic system is activated. In further experiments, the micro(μ)-plasmin–KD1L17R-KT complex inhibited urokinase-induced plasminogen activation on phorbol-12-myristate-13-acetate-stimulated U937 monocyte-like cells, whereas the μ-plasmin–KD1L17R-VT complex failed to inhibit this process. In conclusion, KD1L17R-KT inhibits the active site of plasmin as well as acts as a decoy receptor for the kringle domain(s) of plasminogen/plasmin; hence, it limits both plasmin generation and activity. With its dual function, KD1L17R-KT could serve as a preferred agent for controlling plasminogen activation in pathological processes. PMID:24383758

Decoy plasminogen receptor containing a selective Kunitz-inhibitory domain.

PubMed

Kumar, Yogesh; Vadivel, Kanagasabai; Schmidt, Amy E; Ogueli, Godwin I; Ponnuraj, Sathya M; Rannulu, Nalaka; Loo, Joseph A; Bajaj, Madhu S; Bajaj, S Paul

2014-01-28

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2' residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 μM) but not to KD1L17R-VT. Furthermore, KD1L17R-KT inhibited tPA-induced plasma clot fibrinolysis more efficiently than KD1L17R-VT. Additionally, compared to ε-aminocaproic acid KD1L17R-KT was more effective in reducing blood loss in a mouse liver-laceration injury model, where the fibrinolytic system is activated. In further experiments, the micro(μ)-plasmin-KD1L17R-KT complex inhibited urokinase-induced plasminogen activation on phorbol-12-myristate-13-acetate-stimulated U937 monocyte-like cells, whereas the μ-plasmin-KD1L17R-VT complex failed to inhibit this process. In conclusion, KD1L17R-KT inhibits the active site of plasmin as well as acts as a decoy receptor for the kringle domain(s) of plasminogen/plasmin; hence, it limits both plasmin generation and activity. With its dual function, KD1L17R-KT could serve as a preferred agent for controlling plasminogen activation in pathological processes.

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promotermore » activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells treated with nicotine displayed enhanced invasiveness. ► Nicotine induces uPAR expression and, in turn, stimulates invasiveness. ► MAPK/AP-1 and ROS/NF-κB signals are involved in nicotine-induced uPAR.« less

Soluble Urokinase-Type Plasminogen Activator Receptor in Black Americans with CKD.

PubMed

Luo, Shengyuan; Coresh, Josef; Tin, Adrienne; Rebholz, Casey M; Chen, Teresa K; Hayek, Salim S; Tracy, Melissa; Lipkowitz, Michael S; Appel, Lawrence J; Levey, Andrew S; Inker, Lesley A; Reiser, Jochen; Grams, Morgan Erika

2018-06-14

Black Americans with and without APOL1 kidney disease risk variants face high risk of ESKD. Soluble urokinase-type plasminogen activator receptor (suPAR), a circulating signaling protein and marker of immune activation, constitutes a promising biomarker of CKD-associated risks. We aimed to quantify the associations between serum suPAR concentration and adverse outcomes in Black Americans with and without APOL1 kidney disease risk variants, over and above iodine-125 iothalamate measured GFR and proteinuria. Using data from the African-American Study of Kidney Disease and Hypertension, a multicenter clinical trial followed by a cohort phase with a median total follow-up of 9.7 years (interquartile range, 6.5-10.9 years), we examined the associations of suPAR with CKD progression (defined as doubling of serum creatinine or ESKD), ESKD, worsening proteinuria (defined as pre-ESKD doubling of 24-hour urine protein-to-creatinine ratio to ≥220 mg/g), and all-cause death. At baseline, the median suPAR was 4462 pg/ml, mean measured GFR was 46 ml/min per 1.73 m 2 , and median 24-hour urine protein-to-creatinine ratio was 80 mg/g. After controlling for baseline demographics, randomization arm, GFR, proteinuria, APOL1 risk status, and clinical risk factors, there was a 1.26-times higher risk for CKD progression per SD higher baseline log-transformed suPAR (hazard ratio [HR], 1.26; 95% confidence interval [95% CI], 1.11 to 1.43; P <0.001). Higher suPAR was also independently associated with risk of ESKD (HR, 1.36; 95% CI, 1.17 to 1.58; P <0.001) and death (HR, 1.25; 95% CI, 1.08 to 1.45; P =0.003). suPAR was only associated with worsening proteinuria in patients with two APOLI risk alleles (HR, 1.46; 95% CI, 1.08 to 1.99; P =0.02). Higher suPAR was associated with various adverse outcomes in Black Americans with CKD, with and without APOL1 kidney disease risk variants, independently of proteinuria and GFR. Copyright © 2018 by the American Society of Nephrology.

Galectin-8 induces partial epithelial–mesenchymal transition with invasive tumorigenic capabilities involving a FAK/EGFR/proteasome pathway in Madin–Darby canine kidney cells

PubMed Central

Oyanadel, Claudia; Holmes, Christopher; Pardo, Evelyn; Retamal, Claudio; Shaughnessy, Ronan; Smith, Patricio; Cortés, Priscilla; Bravo-Zehnder, Marcela; Metz, Claudia; Feuerhake, Teo; Romero, Diego; Roa, Juan Carlos; Montecinos, Viviana; Soza, Andrea; González, Alfonso

2018-01-01

Epithelial cells can acquire invasive and tumorigenic capabilities through epithelial–mesenchymal-transition (EMT). The glycan-binding protein galectin-8 (Gal-8) activates selective β1-integrins involved in EMT and is overexpressed by certain carcinomas. Here we show that Gal-8 overexpression or exogenous addition promotes proliferation, migration, and invasion in nontumoral Madin–Darby canine kidney (MDCK) cells, involving focal-adhesion kinase (FAK)-mediated transactivation of the epidermal growth factor receptor (EGFR), likely triggered by α5β1integrin binding. Under subconfluent conditions, Gal-8–overexpressing MDCK cells (MDCK-Gal-8H) display hallmarks of EMT, including decreased E-cadherin and up-regulated expression of vimentin, fibronectin, and Snail, as well as increased β-catenin activity. Changes related to migration/invasion included higher expression of α5β1 integrin, extracellular matrix-degrading MMP13 and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) protease systems. Gal-8–stimulated FAK/EGFR pathway leads to proteasome overactivity characteristic of cancer cells. Yet MDCK-Gal-8H cells still develop apical/basolateral polarity reverting EMT markers and proteasome activity under confluence. This is due to the opposite segregation of Gal-8 secretion (apical) and β1-integrins distribution (basolateral). Strikingly, MDCK-Gal-8H cells acquired tumorigenic potential, as reflected in anchorage-independent growth in soft agar and tumor generation in immunodeficient NSG mice. Therefore, Gal-8 can promote oncogenic-like transformation of epithelial cells through partial and reversible EMT, accompanied by higher proliferation, migration/invasion, and tumorigenic properties. PMID:29298841

Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia. Comparison with the acute respiratory distress syndrome.

PubMed

Günther, A; Mosavi, P; Heinemann, S; Ruppert, C; Muth, H; Markart, P; Grimminger, F; Walmrath, D; Temmesfeld-Wollbrück, B; Seeger, W

2000-02-01

Changes in the alveolar hemostatic balance in severe pneumonia were compared with those in the acute respiratory distress syndrome (ARDS). Analysis was performed in bronchoalveolar lavage fluids (BALF) of patients with ARDS triggered by nonpulmonary underlying events in the absence of lung infection (ARDS; n = 25), pneumonia demanding mechanical ventilation (PNEU-vent; n = 114), spontaneously breathing patients with pneumonia (PNEU-spon; n = 40), and ARDS in combination with lung infection (ARDS+PNEU; n = 43); comparison with healthy control subjects (n = 35) was performed. In all groups of patients, BALF total procoagulant activity was increased by nearly two orders of magnitude, being largely attributable to the tissue factor pathway of coagulation. Concomitantly, markedly reduced overall fibrinolytic capacity (fibrin plate assay) was noted in the lavage fluids of all patients. BALF levels of urokinase-type plasminogen activator were significantly reduced throughout, whereas the lavage concentrations of tissue-type plasminogen activator did not differ from those in control subjects. In addition, markedly enhanced levels of plasminogen activator- inhibitor I and alpha(2)-antiplasmin were noted in ARDS, ARDS+PNEU, and PNEU-vent, but not in PNEU-spon. In all groups of patients, the changes in the lavage enzymatic activities were paralleled by manifold increased BALF concentrations of fibrinopeptide A and D-dimer, reflecting in vivo coagulation processes. Within the overall number of patients with pneumonia, changes in the alveolar hemostatic balance were more prominent in alveolar and interstitial pneumonia than in bronchopneumonia. Acute inflammatory lung injury, whether triggered by nonpulmonary systemic events or primary lung infection, is thus consistently characterized by both enhanced procoagulant and depressed fibrinolytic activities in the alveolar lining layer, with the appearance of fibrin formation in this compartment. Profile and extent of changes in severe pneumonia demanding respirator therapy are virtually identical to those in ARDS, whereas somewhat less prominent alterations of the alveolar hemostatic balance are noted in spontaneously breathing patients with pneumonia.

The evolution of recombinant thrombolytics: Current status and future directions

PubMed Central

Adivitiya; Khasa, Yogender Pal

2017-01-01

ABSTRACT Cardiovascular disorders are on the rise worldwide due to alcohol abuse, obesity, hypertension, raised blood lipids, diabetes and age-related risks. The use of classical antiplatelet and anticoagulant therapies combined with surgical intervention helped to clear blood clots during the inceptive years. However, the discovery of streptokinase and urokinase ushered the way of using these enzymes as thrombolytic agents to degrade the fibrin network with an issue of systemic hemorrhage. The development of second generation plasminogen activators like anistreplase and tissue plasminogen activator partially controlled this problem. The third generation molecules, majorly t-PA variants, showed desirable properties of improved stability, safety and efficacy with enhanced fibrin specificity. Plasmin variants are produced as direct fibrinolytic agents as a futuristic approach with targeted delivery of these drugs using liposome technlogy. The novel molecules from microbial, plant and animal origin present the future of direct thrombolytics due to their safety and ease of administration. PMID:27696935

Fibrin-specific and effective clot lysis requires both plasminogen activators and for them to be in a sequential rather than simultaneous combination.

PubMed

Pannell, R; Li, S; Gurewich, V

2017-08-01

Thrombolysis with tissue plasminogen activator (tPA) has been a disappointment and has now been replaced by an endovascular procedure whenever possible. Nevertheless, thrombolysis remains the only means by which circulation in a thrombosed artery can be restored rapidly. In contrast to tPA monotherapy, endogenous fibrinolysis uses both tPA and urokinase plasminogen activator (uPA), whose native form is a proenzyme, prouPA. This combination is remarkably effective as evidenced by the fibrin degradation product, D-dimer, which is invariably present in plasma. The two activators have complementary mechanisms of plasminogen activation and are synergistic in combination. Since tPA initiates fibrinolysis when released from the vessel wall and prouPA is in the blood, they induce fibrinolysis sequentially. It was postulated that this may be more effective and fibrin-specific. The hypothesis was tested in a model of clot lysis in plasma in which a clot was first exposed to tPA for 5 min, washed and incubated with prouPA. Lysis was compared with that of clots incubated with both activators simultaneously. The sequential combination was almost twice as effective and caused less fibrinogenolysis than the simultaneous combination (p < 0.0001) despite having significantly less tPA, as a result of the wash. A mechanism is described by which this phenomenon can be explained. The findings are believed to have significant therapeutic implications.

The fibrinolytic system: A new target for treatment of depression with psychedelics.

PubMed

Idell, R D; Florova, G; Komissarov, A A; Shetty, S; Girard, R B S; Idell, S

2017-03-01

Current understanding of the neurobiology of depression has grown over the past few years beyond the traditional monoamine theory of depression to include chronic stress, inflammation and disrupted synaptic plasticity. Tissue plasminogen activator (tPA) is a key factor that not only promotes fibrinolysis via the activation of plasminogen, but also contributes to regulation of synaptic plasticity and neurogenesis through plasmin-mediated activation of a probrain derived neurotrophic factor (BDNF) to mature BDNF. ProBDNF activation could potentially be supressed by competition with fibrin for plasmin and tPA. High affinity binding of plasmin and tPA to fibrin could result in a decrease of proBDNF activation during brain inflammation leading to fibrosis further perpetuating depressed mood. There is a paucity of data explaining the possible role of the fibrinolytic system or aberrant extravascular fibrin deposition in depression. We propose that within the brain, an imbalance between tPA and urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) and neuroserpin favors the inhibitors, resulting in changes in neurogenesis, synaptic plasticity, and neuroinflammation that result in depressive behavior. Our hypothesis is that peripheral inflammation mediates neuroinflammation, and that cytokines such as tumor necrosis factor alpha (TNF-α) can inhibit the fibrinolytic system by up- regulating PAI-1 and potentially neuroserpin. We propose that the decrement of the activity of tPA and uPA occurs with downregulation of uPA in part involving the binding and clearance from the surface of neural cells of uPA/PAI-1 complexes by the urokinase receptor uPAR. We infer that current antidepressants and ketamine mitigate depressive symptoms by restoring the balance of the fibrinolytic system with increased activity of tPA and uPA with down-regulated intracerebral expression of their inhibitors. We lastly hypothesize that psychedelic 5-ht2a receptor agonists, such as psilocybin, can improve mood through anti- inflammatory and pro-fibrinolytic effects that include blockade of TNF-α activity leading to decreased PAI-1 activity and increased clearance. The process involves disinhibition of tPA and uPA with subsequent increased cleavage of proBDNF which promotes neurogenesis, decreased neuroinflammation, decreased fibrin deposition, normalized glial-neuronal cross-talk, and optimally functioning neuro-circuits involved in mood. We propose that psilocybin can alleviate deleterious changes in the brain caused by chronic stress leading to restoration of homeostatic brain fibrinolytic capacity leading to euthymia. Copyright © 2017 Elsevier Ltd. All rights reserved.

Enhanced Venous Thrombus Resolution in Plasminogen Activator Inhibitor Type-2 Deficient Mice

PubMed Central

Siefert, Suzanne A; Chabasse, Christine; Mukhopadhyay, Subhradip; Hoofnagle, Mark H; Strickland, Dudley K; Sarkar, Rajabrata; Antalis, Toni M

2014-01-01

Background The resolution of deep vein thrombosis (DVT) requires an inflammatory response and mobilization of proteases, such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs), to degrade the thrombus and remodel the injured vein wall. PAI-2 is a serine protease inhibitor (serpin) with unique immunosuppressive and cell survival properties that was originally identified as an inhibitor of uPA. Objective To investigate the role of PAI-2 in venous thrombus formation and resolution. Methods Venous thrombus resolution was compared in wild type C57BL/6, PAI-2 -/- and PAI-1 -/- mice using the stasis model of DVT. Formed thrombi were harvested, thrombus weights were recorded, and tissue was analyzed for uPA, and MMP activities, PAI-1 expression, and the nature of inflammatory cell infiltration. Results We found that absence of PAI-2 enhanced venous thrombus resolution, while thrombus formation was unaffected. Enhanced venous thrombus resolution in PAI-2 -/- mice was associated with increased uPA activity and reduced levels of PAI-1, with no significant effect on MMP-2 and -9 activities. PAI-1 deficiency resulted in an increase in thrombus resolution similar to PAI-2 deficiency, but additionally reduced venous thrombus formation and altered MMP activity. PAI-2 deficient thrombi had increased levels of the neutrophil chemoattractant, CXCL2, which was associated with early enhanced neutrophil recruitment. Conclusions These data identify PAI-2 as a novel regulator of venous thrombus resolution, which modulates several pathways involving both inflammatory and uPA activity mechanisms, distinct from PAI-1. Further examination of these pathways may lead to potential therapeutic prospects in accelerating thrombus resolution. PMID:25041188

The human airway trypsin-like protease modulates the urokinase receptor (uPAR, CD87) structure and functions.

PubMed

Beaufort, Nathalie; Leduc, Dominique; Eguchi, Hiroshi; Mengele, Karin; Hellmann, Daniela; Masegi, Tsukio; Kamimura, Takashi; Yasuoka, Susumu; Fend, Falko; Chignard, Michel; Pidard, Dominique

2007-05-01

The human airway trypsin-like protease (HAT) is a respiratory epithelium-associated, type II transmembrane serine protease, which is also detected as an extracellular enzyme in lung fluids during airway inflammatory disorders. We have evaluated its capacity to affect the urokinase-type plasminogen activator receptor (uPAR), a membrane glycolipid-anchored, three-domain (D1D2D3) glycoprotein that plays a crucial role in innate immunity and inflammation by supporting cell migration and matrix degradation, with structure and biological properties that can be regulated via limited endoproteolysis. With the use of immunoblotting, flow immunocytometry, and ELISA analyses applied to a recombinant uPAR protein and to uPAR-expressing monocytic and human bronchial epithelial cells, it was shown that exposure of uPAR to soluble HAT in the range of 10-500 nM resulted in the proteolytic processing of the full-length (D1D2D3) into the truncated (D2D3) species, with cleavage occurring in the D1 to D2 linker sequence after arginine residues at position 83 and 89. Using immunohistochemistry, we found that both HAT and uPAR were expressed in the human bronchial epithelium. Moreover, transient cotransfection in epithelial cells showed that membrane coexpression of the two partners produced a constitutive and extensive shedding of the D1 domain, occurring for membrane-associated HAT concentrations in the nanomolar range. Because the truncated receptor was found to be unable to bind two of the major uPAR ligands, the adhesive matrix protein vitronectin and the serine protease urokinase, it thus appears that proteolytic regulation of uPAR by HAT is likely to modulate cell adherence and motility, as well as tissue remodeling during the inflammatory response in the airways.

Network of Surface-Displayed Glycolytic Enzymes in Mycoplasma pneumoniae and Their Interactions with Human Plasminogen

PubMed Central

Gründel, Anne; Pfeiffer, Melanie; Jacobs, Enno

2015-01-01

In different bacteria, primarily cytosolic and metabolic proteins are characterized as surface localized and interacting with different host factors. These moonlighting proteins include glycolytic enzymes, and it has been hypothesized that they influence the virulence of pathogenic species. The presence of surface-displayed glycolytic enzymes and their interaction with human plasminogen as an important host factor were investigated in the genome-reduced and cell wall-less microorganism Mycoplasma pneumoniae, a common agent of respiratory tract infections of humans. After successful expression of 19 glycolytic enzymes and production of polyclonal antisera, the localization of proteins in the mycoplasma cell was characterized using fractionation of total proteins, colony blot, mild proteolysis and immunofluorescence of M. pneumoniae cells. Eight glycolytic enzymes, pyruvate dehydrogenases A to C (PdhA-C), glyceraldehyde-3-phosphate dehydrogenase (GapA), lactate dehydrogenase (Ldh), phosphoglycerate mutase (Pgm), pyruvate kinase (Pyk), and transketolase (Tkt), were confirmed as surface expressed and all are able to interact with plasminogen. Plasminogen bound to recombinant proteins PdhB, GapA, and Pyk was converted to plasmin in the presence of urokinase plasminogen activator and plasmin-specific substrate d-valyl-leucyl-lysine-p-nitroanilide dihydrochloride. Furthermore, human fibrinogen was degraded by the complex of plasminogen and recombinant protein PdhB or Pgm. In addition, surface-displayed proteins (except PdhC) bind to human lung epithelial cells, and the interaction was reduced significantly by preincubation of cells with antiplasminogen. Our results suggest that plasminogen binding and activation by different surface-localized glycolytic enzymes of M. pneumoniae may play a role in successful and long-term colonization of the human respiratory tract. PMID:26667841

Analysis of urine composition in type Ⅱ diabetic mice after intervention therapy using holothurian polypeptides

NASA Astrophysics Data System (ADS)

Li, Yanyan; Xu, Jiajie; Su, Xiurong

2017-07-01

Hydrolysates and peptide fractions (PF) obtained from sea cucumber with commercial enzyme were studied on the hpyerglycemic and renal protective effects on db/db rats using urine metabolomics. Compared with the control group the polypeptides from the two species could significantly reduce the urine glucose and urea. We also tried to address the compositions of highly expressed urinary proteins using a proteomics approach. They were serum albumins, AMBP proteins, negative trypsin, elastase and urinary protein, GAPDH, a receptor of urokinase-type plasminogen activator (uPAR), and Ig kappa chain C region. We used the electronic nose to quickly detect changes in the volatile substances in mice urine after holothurian polypeptides fed, and the results show it can identify the difference between treatment groups with the control group without overlapping. The protein express mechanism of holothurian polypeptides treating diabetes was discussed, and we suggested these two peptides with the hypoglycemic and renal protective activity might be utilized as nutraceuticals.

Contact activation: a revision.

PubMed

Schmaier, A H

1997-07-01

In conclusion, a revised view of the contact system has been presented. This system has little to do with the initiation of hemostasis. Like lupus anticoagulants, deficiencies of contact proteins give prolonged APTTs but may be risk factors for thrombosis. BK from kininogens is a potent modulator of vascular biology inducing vasodilation, tissue plasminogen activator release, and prostacyclin liberation. Kininogens, themselves, are selective inhibitors of alpha-thrombin-induced platelet activation preventing alpha-thrombin from cleaving the cloned thrombin receptor after arginine41. Kininogens' alpha-thrombin inhibitory activity exists in intact kininogens, BK, and all of BK's breakdown products. HK also is the pivotal protein for contact protein assembly on endothelium. It is the receptor for prekallikrein which when bound to HK becomes activated to kallikrein by an endothelial cell enzyme system independent of activated forms of plasma factor XII. Prekallikrein activation on endothelial cells results in kinetically favorable single chain urokinase and plasminogen activation. Thus the "physiologic, negatively charged surface" for contact system activation is really the assembly of these proteins on cell membranes and activation by membrane-associated enzymes.

Number and brightness image analysis reveals ATF-induced dimerization kinetics of uPAR in the cell membrane

PubMed Central

Hellriegel, Christian; Caiolfa, Valeria R.; Corti, Valeria; Sidenius, Nicolai; Zamai, Moreno

2011-01-01

We studied the molecular forms of the GPI-anchored urokinase plasminogen activator receptor (uPAR-mEGFP) in the human embryo kidney (HEK293) cell membrane and demonstrated that the binding of the amino-terminal fragment (ATF) of urokinase plasminogen activator is sufficient to induce the dimerization of the receptor. We followed the association kinetics and determined precisely the dimeric stoichiometry of uPAR-mEGFP complexes by applying number and brightness (N&B) image analysis. N&B is a novel fluctuation-based approach for measuring the molecular brightness of fluorophores in an image time sequence in live cells. Because N&B is very sensitive to long-term temporal fluctuations and photobleaching, we have introduced a filtering protocol that corrects for these important sources of error. Critical experimental parameters in N&B analysis are illustrated and analyzed by simulation studies. Control experiments are based on mEGFP-GPI, mEGFP-mEGFP-GPI, and mCherry-GPI, expressed in HEK293. This work provides a first direct demonstration of the dimerization of uPAR in live cells. We also provide the first methodological guide on N&B to discern minor changes in molecular composition such as those due to dimerization events, which are involved in fundamental cell signaling mechanisms.—Hellriegel, C., Caiolfa, V. R., Corti, V., Sidenius, N., Zamai, M. Number and brightness image analysis reveals ATF-induced dimerization kinetics of uPAR in the cell membrane. PMID:21602447

The relationship between levels of plasma-soluble urokinase plasminogen activator receptor (suPAR) and presence of migraine attack and aura.

PubMed

Yılmaz, Nigar; Yılmaz, Mustafa; Sirin, Burcu; Yılmaztekin, Sureyya; Kutlu, Gülnihal

2017-10-01

Migraine is one of the most common types of pain associated with sterile inflammatory conditions. Soluble urokinase plasminogen activator receptor (suPAR) is a potential novel inflammatory marker. We aim to determine the association between serum values of suPAR, procalcitonin, fibrinogen, and high-sensitivity C-reactive protein (hs-CRP) and migraine disease characteristics. The study involved a total of 60 migraine patients (33 patients in the interictal period, 27 patients in the attack period) and 30 healthy individuals. The serum values of suPAR were found to be significantly higher in migraine patients in the attack period than in migraine patients in the interictal period, and in healthy individuals (p < .01 for both). In addition, levels of suPAR were determined to be higher in migraine with aura patients than in migraine without aura patients. When we subdivided migraine patients according to frequency of attack (attacks/month), significant differences were found between the suPAR and procalcitonin levels (measured during the attack period) of those in the frequent-attack group (4-5 or more) versus those in the less frequent attack group (less than 4). Serum levels of procalcitonin were shown to be significantly higher in migraine patients during the attack period compared with migraine patients in the interictal period and in control subjects (p = .001 for both). Significant differences were found between plasma levels of fibrinogen in migraine patients versus control subjects (p < .01). No statistically significant difference was found between levels of hs-CRP in migraine patients versus the control group. These findings may show that presenting a high level of suPAR in migraine patients with attack and aura results to predisposition to occurring on the symptoms and that high levels of suPAR, procalcitonin and fibrinogen in patients with migraine result in neurogenic inflammation during migraine headaches.

Podocyte injury: the role of proteinuria, urinary plasminogen, and oxidative stress

PubMed Central

Tian, Runxia; Wong, Jenny S.; He, John C.; Campbell, Kirk N.

2016-01-01

Podocytes are the key target for injury in proteinuric glomerular diseases that result in podocyte loss, progressive focal segmental glomerular sclerosis (FSGS), and renal failure. Current evidence suggests that the initiation of podocyte injury and associated proteinuria can be separated from factors that drive and maintain these pathogenic processes leading to FSGS. In nephrotic urine aberrant glomerular filtration of plasminogen (Plg) is activated to the biologically active serine protease plasmin by urokinase-type plasminogen activator (uPA). In vivo inhibition of uPA mitigates Plg activation and development of FSGS in several proteinuric models of renal disease including 5/6 nephrectomy. Here, we show that Plg is markedly increased in the urine in two murine models of proteinuric kidney disease associated with podocyte injury: Tg26 HIV-associated nephropathy and the Cd2ap−/− model of FSGS. We show that human podocytes express uPA and three Plg receptors: uPAR, tPA, and Plg-RKT. We demonstrate that Plg treatment of podocytes specifically upregulates NADPH oxidase isoforms NOX2/NOX4 and increases production of mitochondrial-dependent superoxide anion (O2−) that promotes endothelin-1 synthesis. Plg via O2− also promotes expression of the B scavenger receptor CD36 and subsequent increased intracellular cholesterol uptake resulting in podocyte apoptosis. Taken together, our findings suggest that following disruption of the glomerular filtration barrier at the onset of proteinuric disease, podocytes are exposed to Plg resulting in further injury mediated by oxidative stress. We suggest that chronic exposure to Plg could serve as a “second hit” in glomerular disease and that Plg is potentially an attractive target for therapeutic intervention. PMID:27335373

Node-Negative Breast Cancer: Which Patients Should Be Treated?

PubMed Central

Schmidt, Marcus

2008-01-01

Summary Adjuvant systemic therapy has led to markedly improved outcome in early-stage breast cancer. However, the absolute gains from chemotherapy might be modest in node-negative patients. Adjuvant chemotherapy is the only option for triple-negative breast cancer patients and should be used with trastuzumab in HER2-positive patients. Considering the large group of patients with some degree of endocrine responsiveness, adding chemotherapy according to risk is an option. At present, we guide our therapeutic decisions using clinicopathologic risk classifications like the St. Gallen risk category or Adjuvant! online. A downside of these risk estimations is a low specificity and consequently the risk for overtreatment of a considerable number of patients. To spare patients unnecessary toxicities we need more reliable prognostic factors or tumor markers. From the plethora of tumor markers, only urokinase-type plasminogen activator (uPA)/plasminogen activator inhibitor 1 (PAI-1) and certain multiparameter gene expression assays are recommended by the American Society of Clinical Oncology. These tumor markers are presently investigated in clinical trials in node-negative breast cancer (NNBC-3, MINDACT, TAILORx). These studies will hopefully allow us to quantify the risk of progression in the individual patient and to tailor treatment accordingly. This should lead to a more personalized treatment recommendation. PMID:21076603

Generation of Novel Chimeric Mice with Humanized Livers by Using Hemizygous cDNA-uPA/SCID Mice

PubMed Central

Tateno, Chise; Kawase, Yosuke; Tobita, Yoshimi; Hamamura, Satoko; Ohshita, Hiroki; Yokomichi, Hiroshi; Sanada, Harumi; Kakuni, Masakazu; Shiota, Akira; Kojima, Yuha; Ishida, Yuji; Shitara, Hiroshi; Wada, Naoko A.; Tateishi, Hiromi; Sudoh, Masayuki; Nagatsuka, Shin-ichiro; Jishage, Kou-ichi; Kohara, Michinori

2015-01-01

We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression—not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for producing chimeric mice for use in future long-term studies, including hepatitis virus infection analysis or drug toxicity studies. PMID:26536627

Generation of Novel Chimeric Mice with Humanized Livers by Using Hemizygous cDNA-uPA/SCID Mice.

PubMed

Tateno, Chise; Kawase, Yosuke; Tobita, Yoshimi; Hamamura, Satoko; Ohshita, Hiroki; Yokomichi, Hiroshi; Sanada, Harumi; Kakuni, Masakazu; Shiota, Akira; Kojima, Yuha; Ishida, Yuji; Shitara, Hiroshi; Wada, Naoko A; Tateishi, Hiromi; Sudoh, Masayuki; Nagatsuka, Shin-Ichiro; Jishage, Kou-Ichi; Kohara, Michinori

2015-01-01

We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression-not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for producing chimeric mice for use in future long-term studies, including hepatitis virus infection analysis or drug toxicity studies.

A reassessment of soluble urokinase-type plasminogen activator receptor in glomerular disease

PubMed Central

Spinale, Joann M.; Mariani, Laura H.; Kapoor, Shiv; Zhang, Jidong; Weyant, Robert; Song, Peter X.; Wong, Hetty N.; Troost, Jonathan P.; Gadegbeku, Crystal A.; Gipson, Debbie S.; Kretzler, Matthias; Nihalani, Deepak; Holzman, Lawrence B.

2014-01-01

It has been suggested that soluble urokinase receptor (suPAR) is a causative circulating factor for and a biomarker of focal and segmental glomerulosclerosis (FSGS). Here we undertook validation of these assumptions in both mouse and human models. Injection of recombinant suPAR in wild-type mice did not induce proteinuria within 24 hours. Moreover, a disease phenotype was not seen in an inducible transgenic mouse model that maintained elevated suPAR concentrations for 6 weeks. Plasma and urine suPAR concentrations were evaluated as clinical biomarkers in 241 patients with glomerular disease from the prospective, longitudinal multi-center observational NEPTUNE cohort. The serum suPAR concentration at baseline inversely correlated with estimated glomerular filtration rate (eGFR) and the urine suPAR/creatinine ratio positively correlated with the urine protein/creatinine ratio. After adjusting for eGFR and urine protein, neither the serum nor urine suPAR level was an independent predictor of FSGS histopathology. A multivariable mixed-effects model of longitudinal data evaluated the association between the change in serum suPAR concentration from baseline with eGFR. After adjusting for baseline suPAR concentration, age, gender, proteinuria and time, the change in suPAR from baseline was associated with eGFR, but this association was not different for patients with FSGS as compared to other diagnoses. Thus, these results do not support a pathological role for suPAR in FSGS. PMID:25354239

A reassessment of soluble urokinase-type plasminogen activator receptor in glomerular disease.

PubMed

Spinale, Joann M; Mariani, Laura H; Kapoor, Shiv; Zhang, Jidong; Weyant, Robert; Song, Peter X; Wong, Hetty N; Troost, Jonathan P; Gadegbeku, Crystal A; Gipson, Debbie S; Kretzler, Matthias; Nihalani, Deepak; Holzman, Lawrence B

2015-03-01

It has been suggested that soluble urokinase receptor (suPAR) is a causative circulating factor for and a biomarker of focal and segmental glomerulosclerosis (FSGS). Here we undertook validation of these assumptions in both mouse and human models. Injection of recombinant suPAR in wild-type mice did not induce proteinuria within 24 h. Moreover, a disease phenotype was not seen in an inducible transgenic mouse model that maintained elevated suPAR concentrations for 6 weeks. Plasma and urine suPAR concentrations were evaluated as clinical biomarkers in 241 patients with glomerular disease from the prospective, longitudinal multicenter observational NEPTUNE cohort. The serum suPAR concentration at baseline inversely correlated with estimated glomerular filtration rate (eGFR) and the urine suPAR/creatinine ratio positively correlated with the urine protein/creatinine ratio. After adjusting for eGFR and urine protein, neither the serum nor urine suPAR level was an independent predictor of FSGS histopathology. A multivariable mixed-effects model of longitudinal data evaluated the association between the change in serum suPAR concentration from baseline with eGFR. After adjusting for baseline suPAR concentration, age, gender, proteinuria, and time, the change in suPAR from baseline was associated with eGFR, but this association was not different for patients with FSGS as compared with other diagnoses. Thus these results do not support a pathological role for suPAR in FSGS.

Hepatocyte growth factor (HGF) modulates GABAergic inhibition and seizure susceptibility

PubMed Central

Bae, Mihyun H.; Bissonette, Gregory B.; Mars, Wendy M.; Michalopoulos, George K.; Achim, Cristian L.; Depireux, Didier A.; Powell, Elizabeth M.

2009-01-01

Disrupted ontogeny of forebrain inhibitory interneurons leads to neurological disorders, including epilepsy. Adult mice lacking the urokinase plasminogen activator receptor (Plaur) have decreased numbers of neocortical GABAergic interneurons and spontaneous seizures, attributed to a reduction of hepatocyte growth factor/scatter factor (HGF/SF). We report that by increasing endogenous HGF/SF concentration in the postnatal Plaur null mouse brain maintains the interneuron populations in the adult, reverses the seizure behavior and stabilizes the spontaneous electroencephalogram activity. The perinatal intervention provides a pathway to reverse potential birth defects and ameliorate seizures in the adult. PMID:19853606

Protease nexin 1 is a potent urinary plasminogen activator inhibitor in the presence of collagen type IV.

PubMed

Crisp, Robert J; Knauer, Mary F; Knauer, Daniel J

2002-12-06

Protease nexin 1 (PN1) in solution forms inhibitory complexes with thrombin or urokinase, which have opposing effects on the blood coagulation cascade. An initial report provided data supporting the idea that PN1 target protease specificity is under the influence of collagen type IV (1). Although collagen type IV demonstrated no effect on the association rate between PN1 and thrombin, the study reported that the association rate between PN1 and urokinase was allosterically reduced 10-fold. This has led to the generally accepted idea that the primary role of PN1 in the brain is to act as a rapid thrombin inhibition and clearance mechanism during trauma and loss of vascular integrity. In studies to identify the structural determinants of PN1 that mediate the allosteric interaction with collagen type IV, we found that protease specificity was only affected after transient exposure of PN1 to acidic conditions that mimic the elution protocol from a monoclonal antibody column. Because PN1 used in previous studies was purified over a monoclonal antibody column, we propose that the allosteric regulation of PN1 target protease specificity by collagen type IV is a result of the purification protocol. We provide both biochemical and kinetic data to support this conclusion. This finding is significant because it implies that PN1 may play a much larger role in the modeling and remodeling of brain tissues during development and is not simply an extravasated thrombin clearance mechanism as previously suggested.

Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase.

PubMed

Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Liu, Ming C; Shetty, Sreerama

2010-02-01

The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1-100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.

Clinical evaluation of multiple inflammation biomarkers for diagnosis and prognosis for patients with systemic inflammatory response syndrome.

PubMed

Reichsoellner, M; Raggam, R B; Wagner, J; Krause, R; Hoenigl, M

2014-11-01

A multiplexed biomarker bundle consisting of nine different inflammation markers was evaluated regarding their diagnostic and prognostic performances in 159 adult systemic inflammatory response syndrome (SIRS) patients enrolled at the emergency department. Fibronectin, interleukin-8 (IL-8), biotin, and neutrophil gelatinase-associated lipocalin (NGAL) were the most robust markers but were not superior to the already established markers IL-6, C-reactive protein (CRP), procalcitonin (PCT), and soluble urokinase plasminogen activator receptor (suPAR). Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The Soluble Plasminogen Activator Receptor as a Biomarker on Monitoring the Therapy Progress of Pulmonary TB-AFB(+) Patients

PubMed Central

Mardining Raras, Tri Yudani; Noor Chozin, Iin

2010-01-01

The role of soluble soluble urokinase-type plasminogen activator receptor (suPAR) as a biological marker for TB treatment efficacy on active pulmonary TB-AFB(+) patients was investigated. Twenty pulmonary TB-AFB(+) patients participated in a cohort study for six months. The plasma suPAR level was measured using ELISA method before treatment, two months, four months and six months after treatment. At the same time clinical parameters were also measured. Results indicated that all patients (n = 20) showed highest plasma suPAR levels before treatment (median 12.775 ng/mL) and significantly decreased ( P = .0001<.05, R 2 = .890) after 2 months (median 8.019 ng/mL) and 4 months (median 5.771 ng/mL) of treatment, respectively. However, only slightly declined after 6 months therapy (median 5.009 ng/mL), near control group level (median 4.772 ng/mL). Interestingly, the significant reduced of suPAR level was parallel to treatment efficacy and correlated with other clinical and laboratory parameters, that is, decreasing of patients' complaints, increasing of BMI (r = −0.281), thoracic imaging improvement, sputum conversion, decreasing of ESR (r = 0.577) and monocytes count (r = 0.536) with exception the width of lesion in thoracic imaging. In conclusion, the suPAR level in could reflect the progress of TB therapy. PMID:22567258

Real-time assessment of breast surgical margins with fluorescence-guided microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Iftimia, Nicusor V.; Park, Jesung; Maguluri, Gopi N.; Krishnamurthy, Savitri

2017-02-01

A novel multimodal optical imaging approach for real-time assessment of surgical margins on breast cancer lumpectomy specimens is presented. Our approach is to target cancer cells using an optically silent peptide substrate containing two (NIR) fluorochromes, internally quenched, which are cleaved by highly expressed breast cancer enzymes, like urokinase-type plasminogen activator (uPA). Thus this agent becomes highly fluorescent only on the cancer area when the specimen is excited by a NIR laser beam. A fluorescence imager is used to highlight cancer-suspect margins on the surgical specimen, while high-resolution optical coherence tomography (OCT) imaging is used to visualize tissue morphology on the highlighted areas and confirm or rule out cancer presence. This technology will hopefully increase the success rate of cancer surgeries, reduce the risk of cancer recurrence and significantly reduce US healthcare costs.

Tumor therapy with a urokinase plasminogen activator-activated anthrax lethal toxin alone and in combination with paclitaxel.

PubMed

Wein, Alexander N; Liu, Shihui; Zhang, Yi; McKenzie, Andrew T; Leppla, Stephen H

2013-02-01

PA-U2, an engineered anthrax protective antigen that is activated by urokinase was combined with wildtype lethal factor in the treatment of Colo205 colon adenocarcinoma in vitro and B16-BL6 mouse melanoma in vitro and in vivo. This therapy was also tested in combination with the small molecule paclitaxel, based on prior reports suggesting synergy between ERK1/2 inhibition and chemotherapeutics. Colo205 was sensitive to PA-U2/LF while B16-BL6 was not. For the combination treatment of B16-BL6, paclitaxel showed a dose response in vitro, but cells remained resistant to PA-U2/LF even in the presence of paclitaxel. In vivo, each therapy slowed tumor progression, and an additive effect between the two was observed. Since LF targets tumor vasculature while paclitaxel is an antimitotic, it is possible the agents were acting against different cells in the stroma, precluding a synergistic effect. The engineered anthrax toxin PA-U2/LF warrants further development and testing, possibly in combination with an antiangiogenesis therapy such as sunitinib or sorafinib.

Plasminogen-induced aggregation of PANC-1 cells requires conversion to plasmin and is inhibited by endogenous plasminogen activator inhibitor-1.

PubMed

Deshet, Naamit; Lupu-Meiri, Monica; Espinoza, Ingrid; Fili, Oded; Shapira, Yuval; Lupu, Ruth; Gershengorn, Marvin C; Oron, Yoram

2008-09-01

PANC-1 cells express proteinase-activated receptors (PARs)-1, -2, and respond to their activation by transient elevation of cytosolic [Ca(2+)] and accelerated aggregation (Wei et al., 2006, J Cell Physiol 206:322-328). We studied the effect of plasminogen (PGN), an inactive precursor of the PAR-1-activating protease, plasmin (PN) on aggregation of pancreatic adenocarcinoma (PDAC) cells. A single dose of PGN time- and dose-dependently promoted PANC-1 cells aggregation in serum-free medium, while PN did not. PANC-1 cells express urokinase plasminogen activator (uPA), which continuously converted PGN to PN. This activity and PGN-induced aggregation were inhibited by the uPA inhibitor amiloride. PGN-induced aggregation was also inhibited by alpha-antiplasmin and by the PN inhibitor epsilon-aminocaproic acid (EACA). Direct assay of uPA activity revealed very low rate, markedly enhanced in the presence of PGN. Moreover, in PGN activator inhibitor 1-deficient PANC-1 cells, uPA activity and PGN-induced aggregation were markedly potentiated. Two additional human PDAC cell lines, MiaPaCa and Colo347, were assayed for PGN-induced aggregation. Both cell lines responded by aggregation and exhibited PGN-enhanced uPA activity. We hypothesized that the continuous conversion of PGN to PN by endogenous uPA is limited by PN's degradation and negatively controlled by endogenously produced PAI-1. Indeed, we found that PANC-1 cells inactivate PN with t1/2 of approximately 7 h, while the continuous addition of PN promoted aggregation. Our data suggest that PANC-1 cells possess intrinsic, PAI-1-sensitive mechanism for promotion of aggregation and differentiation by prolonged exposure to PGN and, possibly, additional precursors of PARs agonists.

Role of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in psychological stress and depression.

PubMed

Tsai, Shih-Jen

2017-12-22

Major depressive disorder is a common illness worldwide, but the pathogenesis of the disorder remains incompletely understood. The tissue-type plasminogen activator-plasminogen proteolytic cascade is highly expressed in the brain regions involved in mood regulation and neuroplasticity. Accumulating evidence from animal and human studies suggests that tissue-type plasminogen activator and its chief inhibitor, plasminogen activator inhibitor-1, are related to stress reaction and depression. Furthermore, the neurotrophic hypothesis of depression postulates that compromised neurotrophin brain-derived neurotrophic factor (BDNF) function is directly involved in the pathophysiology of depression. In the brain, the proteolytic cleavage of proBDNF, a BDNF precursor, to mature BDNF through plasmin represents one mechanism that can change the direction of BDNF action. We also discuss the implications of tissue-type plasminogen activator and plasminogen activator inhibitor-1 alterations as biomarkers for major depressive disorder. Using drugs that increase tissue-type plasminogen activator or decrease plasminogen activator inhibitor-1 levels may open new avenues to develop conceptually novel therapeutic strategies for depression treatment.

TMPRSS4 induces cancer cell invasion through pro-uPA processing.

PubMed

Min, Hye-Jin; Lee, Myung Kyu; Lee, Jung Weon; Kim, Semi

2014-03-28

TMPRSS4 is a novel type II transmembrane serine protease that is highly expressed on the cell surface in pancreatic, thyroid, colon, and other cancer tissues. Previously, we demonstrated that TMPRSS4 mediates cancer cell invasion, epithelial-mesenchymal transition, and metastasis and that increased TMPRSS4 expression correlates with colorectal cancer progression. We also demonstrated that TMPRSS4 upregulates urokinase-type plasminogen activator (uPA) gene expression to induce cancer cell invasion. However, it remains unknown how proteolytic activity of TMPRSS4 contributes to invasion. In this study, we report that TMPRSS4 directly converted inactive pro-uPA into the active form through its proteolytic activity. Analysis of conditioned medium from cells overexpressing TMPRSS4 demonstrated that the active TMPRSS4 protease domain is released from the cells and is associated with the plasma membrane. Furthermore, TMPRSS4 could increase pro-uPA-mediated invasion in a serine proteolytic activity-dependent manner. These observations suggest that TMPRSS4 is an upstream regulator of pro-uPA activation. This study provides valuable insights into the proteolytic function of TMPRSS4 as well as mechanisms for the control of invasion. Copyright © 2014 Elsevier Inc. All rights reserved.

Binding of anti-SSA antibodies to apoptotic fetal cardiocytes stimulates urokinase plasminogen activator (uPA)/uPA receptor-dependent activation of TGF-β and potentiates fibrosis.

PubMed

Briassouli, Paraskevi; Rifkin, Daniel; Clancy, Robert M; Buyon, Jill P

2011-11-15

In congenital heart block (CHB), binding of maternal anti-SSA/Ro Abs to fetal apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases urokinase plasminogen activator (uPA)/uPA receptor (uPAR)-dependent plasmin activation. Because the uPA/uPAR system plays a role in TGF-β activation, we evaluated whether anti-Ro binding to apoptotic cardiocytes enhances plasmin-mediated activation of TGF-β, thereby promoting a profibrosing phenotype. Supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes bound by IgG from a mother whose child had CHB (apoptotic-CHB-IgG [apo-CHB-IgG]) exhibited significantly increased levels of active TGF-β compared with supernatants from cocultures of healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor. Treatment of the culture medium with anti-TGF-β Ab or TGF-β inhibitor (SB431542) abrogated the luciferase response, thereby confirming TGF-β dependency. Increased uPA levels and activity were present in supernatants generated from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes compared with healthy cardiocytes and apoptotic cardiocytes preincubated with IgG from a healthy donor, respectively. Treatment of apo-CHB-IgG cardiocytes with anti-uPAR or anti-uPA Abs or plasmin inhibitor aprotinin prior to coculturing with healthy cardiocytes attenuated TGF-β activation. Supernatants derived from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes promoted Smad2 phosphorylation and fibroblast transdifferentiation, as evidenced by increased smooth muscle actin and collagen expression, which decreased when fibroblasts were treated with supernatants from cocultures pretreated with uPAR Abs. These data suggested that binding of anti-Ro Abs to apoptotic cardiocytes triggers TGF-β activation, by virtue of increasing uPAR-dependent uPA activity, thus initiating and amplifying a cascade of events that promotes myofibroblast transdifferentiation and scar.

Effects of TGF-β1 on plasminogen activation in human dental pulp cells: Role of ALK5/Smad2, TAK1 and MEK/ERK signalling.

PubMed

Chang, Mei-Chi; Chang, Hsiao-Hua; Lin, Po-Shuan; Huang, Yu-An; Chan, Chiu-Po; Tsai, Yi-Ling; Lee, Shen-Yang; Jeng, Po-Yuan; Kuo, Han-Yueh; Yeung, Sin-Yuet; Jeng, Jiiang-Huei

2018-04-01

Transforming growth factor-β1 (TGF-β1) plays an important role in the pulpal repair and dentinogenesis. Plasminogen activation (PA) system regulates extracellular matrix turnover. In this study, we investigated the effects of TGF-β1 on PA system of dental pulp cells and its signalling pathways. Dental pulp cells were treated with different concentrations of TGF-β1. MTT assay, reverse transcription-polymerase chain reaction, Western blotting and enzyme-linked immunosorbant assay (ELISA) were used to detect the effect of TGF-β1 on cell viability, mRNA and protein expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) as well as their secretion. The phosphorylation of Smad2 and TAK1 was analysed by Pathscan ELISA or Western blotting. Cells were pretreated with SB431542 (ALK5/Smad2/3 inhibitor), 5z-7-oxozeaenol (TAK1 inhibitor) and U0126 (MEK/ERK inhibitor) for examining the related signalling. TGF-β1 slightly inhibited cell growth that was reversed by SB431542. TGF-β1 upregulated both RNA and protein expression of PAI-1 and uPAR, whereas it downregulated uPA expression. Accordingly, TGF-β1 stimulated PAI-1 and soluble uPAR (suPAR) secretion of pulp cells, whereas uPA secretion was inhibited. TGF-β1 induced the phosphorylation of Smad2 and TAK1. In addition, SB431542, 5z-7-oxozeaenol and U0126 attenuated the TGF-β1-induced secretion of PAI-1 and suPAR. These results indicate that TGF-β1 is possibly involved in the repair/regeneration and inflammatory processes of dental pulp via regulation of PAI-1, uPA and uPAR. These effects of TGF-β1 are related to activation of ALK5/Smad2, TAK1 and MEK/ERK signalling pathways. Clarifying the signal transduction for the effects of TGF-β1 is helpful for pulpo-dentin regeneration and tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

The urokinase plasminogen activator system components are regulated by vascular endothelial growth factor D in bovine oviduct.

PubMed

García, Daniela C; Russo-Maenza, Agostina; Miceli, Dora C; Valdecantos, Pablo A; Roldán-Olarte, Mariela

2018-06-08

SummaryThe mammalian oviduct plays a pivotal role in the success of early reproductive events. The urokinase plasminogen activator system (uPAS) is present in the bovine oviduct and is involved in extracellular matrix remodelling through plasmin generation. This system can be regulated by several members of the vascular endothelial growth factors (VEGF) and their receptors. In this study, the VEGF-D effect on the regulation of uPAS was evaluated. First, RT-polymerase chain reaction (PCR) analyses were used to evidence the expression of VEGF-D and its receptors in oviductal epithelial cells (BOEC). VEGF-D, VEGFR2 and VEGFR3 transcripts were found in ex vivo and in vitro BOEC, while only VEGFR2 mRNA was present after in vitro conditions. VEGF-D showed a regulatory effect on uPAS gene expression in a dose-dependent manner, inducing an increase in the expression of both uPA and its receptor (uPAR) at 24 h post-induction and decreases in the expression of its inhibitor (PAI-1). In addition, the regulation of cell migration induced by VEGF-D and uPA in BOEC monolayer cultures was analyzed. The wound areas of monolayer cultures incubated with VEGF-D 10 ng/ml or uPA 10 nM were modified and significant differences were found at 24 h for both stimulations. These results indicated that uPAS and VEGF-D systems can modify the arrangement of the bovine oviductal epithelium and contribute to the correct maintenance of the oviductal microenvironment.

Molecular mechanism inhibiting human hepatocarcinoma cell invasion by 6-shogaol and 6-gingerol.

PubMed

Weng, Chia-Jui; Chou, Chai-Ping; Ho, Chi-Tang; Yen, Gow-Chin

2012-08-01

We previously demonstrated that 6-shogaol and 6-gingerol, two active compounds in ginger (Zingiber officinale), possess antiinvasive activity against highly metastatic hepatoma cells. The aims of this study were to evaluate the inhibitory effect and molecular mechanism underlying the transcription and translation of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) in Hep3B cells as well as the antiangiogenic activity of 6-gingerol and 6-shogaol. By gelatin zymography and luciferase reporter gene assays, we found that 6-gingerol and 6-shogaol regulate MMP-2/-9 transcription. Moreover, 6-gingerol directly decreased expression of uPA, but the 6-shogaol-mediated decrease in uPA was accompanied by up-regulation of plasminogen activator inhibitor (PAI)-1. 6-Gingerol and 6-shogaol concentrations of ≥ 10 μM and ≥ 2.5 μM, respectively, significantly inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) and PI3K/Akt signaling, the activation of NF-κB, and the translocation of NF-κB and STAT3. Incubation of 6-gingerol or 6-shogaol with human umbilical vein endothelial cells or rat aortas significantly attenuated tube formation. 6-Shogaol and 6-gingerol effectively inhibit invasion and metastasis of hepatocellular carcinoma through diverse molecular mechanisms, including inhibition of the MAPK and PI3k/Akt pathways and NF-κB and STAT3 activities to suppress expression of MMP-2/-9 and uPA and block angiogenesis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dimerization controls the lipid raft partitioning of uPAR/CD87 and regulates its biological functions

PubMed Central

Cunningham, Orla; Andolfo, Annapaola; Santovito, Maria Lisa; Iuzzolino, Lucia; Blasi, Francesco; Sidenius, Nicolai

2003-01-01

The urokinase-type plasminogen activator receptor (uPAR/CD87) is a glycosylphosphatidylinositol-anchored membrane protein with multiple functions in extracellular proteolysis, cell adhesion, cell migration and proliferation. We now report that cell surface uPAR dimerizes and that dimeric uPAR partitions preferentially to detergent-resistant lipid rafts. Dimerization of uPAR did not require raft partitioning as the lowering of membrane cholesterol failed to reduce dimerization and as a transmembrane uPAR chimera, which does not partition to lipid rafts, also dimerized efficiently. While uPA bound to uPAR independently of its membrane localization and dimerization status, uPA-induced uPAR cleavage was strongly accelerated in lipid rafts. In contrast to uPA, the binding of Vn occurred preferentially to raft- associated dimeric uPAR and was completely blocked by cholesterol depletion. PMID:14609946

Urokinase–urokinase receptor interaction mediates an inhibitory signal for HIV-1 replication

PubMed Central

Alfano, Massimo; Sidenius, Nicolai; Panzeri, Barbara; Blasi, Francesco; Poli, Guido

2002-01-01

Elevated levels of soluble urokinase-type plasminogen activator (uPA) receptor, CD87/u-PAR, predict survival in individuals infected with HIV-1. Here, we report that pro-uPA (or uPA) inhibits HIV-1 expression in U937-derived chronically infected promonocytic U1 cells stimulated with phorbol 12-myristate 13-acetate (PMA) or tumor necrosis factor-α (TNF-α). However, pro-uPA did not inhibit PMA or TNF-α-dependent activation of nuclear factor-kB or activation protein-1 in U1 cells. Cell-associated HIV protein synthesis also was not decreased by pro-uPA, although the release of virion-associated reverse transcriptase activity was substantially inhibited, suggesting a functional analogy between pro-uPA and the antiviral effects of IFNs. Indeed, cell disruption reversed the inhibitory effect of pro-uPA on activated U1 cells, and ultrastructural analysis confirmed that virions were preferentially retained within cell vacuoles in pro-uPA treated cells. Neither expression of endogenous IFNs nor activation of the IFN-inducible Janus kinase/signal transducer and activator of transcription pathway were induced by pro-uPA. Pro-uPA also inhibited acute HIV replication in monocyte-derived macrophages and activated peripheral blood mononuclear cells, although with great inter-donor variability. However, pro-uPA inhibited HIV replication in acutely infected promonocytic U937 cells and in ex vivo cultures of lymphoid tissue infected in vitro. Because these effects occurred at concentrations substantially lower than those affecting thrombolysis, pro-uPA may represent a previously uncharacterized class of antiviral agents mimicking IFNs in their inhibitory effects on HIV expression and replication. PMID:12084931

Successful Tissue Plasminogen Activator for a Patient with Stroke After Stanford Type A Aortic Dissection Treatment.

PubMed

Matsuzono, Kosuke; Suzuki, Masayuki; Arai, Naoto; Kim, Younhee; Ozawa, Tadashi; Mashiko, Takafumi; Shimazaki, Haruo; Koide, Reiji; Fujimoto, Shigeru

2018-07-01

Some stroke patients with the acute aortic dissection receiving thrombolysis treatment resulted in fatalities. Thus, the concurrent acute aortic dissection is the contraindication for the intravenous recombinant tissue-type plasminogen activator. However, the safety and the effectiveness of the intravenous recombinant tissue-type plasminogen activator therapy are not known in patients with stroke some days after acute aortic dissection treatment. Here, we first report a case of a man with a cardioembolism due to the nonvalvular atrial fibrillation, who received the intravenous recombinant tissue-type plasminogen activator therapy 117 days after the traumatic Stanford type A acute aortic dissection operation. Without the intravenous recombinant tissue-type plasminogen activator therapy, the prognosis was expected to be miserable. However, the outcome was good with no complication owing to the intravenous recombinant tissue-type plasminogen activator therapy. Our case suggests the effectiveness and the safety of the intravenous recombinant tissue-type plasminogen activator therapy to the ischemic stroke some days after acute aortic dissection treatment. Copyright © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved.

Insights into abnormal hemostasis in the Quebec platelet disorder from analyses of clot lysis.

PubMed

Diamandis, M; Adam, F; Kahr, W H A; Wang, P; Chorneyko, K A; Arsenault, A L; Rivard, G E; Hayward, C P M

2006-05-01

The Quebec platelet disorder (QPD) is inherited and characterized by delayed-onset bleeding following hemostatic challenge. Other characteristics include increased expression and storage of active urokinase-type plasminogen activator (u-PA) in platelets in the setting of normal to increased u-PA in plasma. There is also consumption of platelet plasminogen activator inhibitor-1 and increased generation of plasmin in platelets accompanied by proteolysis of stored alpha-granule proteins, including Factor V. Although fibrinolysis has been proposed to contribute to QPD bleeding, the effects of QPD blood and platelets on clot lysis have not been evaluated. We used thromboelastography (TEG), biochemical evaluations of whole blood clot lysis, assessments of clot ultrastructure, and perfusion of blood over preformed fibrin to gain insights into the disturbed hemostasis in the QPD. Thromboelastography was not sensitive to the increased u-PA in QPD blood. However, there was abnormal plasmin generation in QPD whole blood clots, generated at low shear, with biochemical evidence of increased fibrinolysis. The incorporation of QPD platelets into a forming clot led to progressive disruption of fibrin and platelet aggregates unless drugs were added to inhibit plasmin. In whole blood perfusion studies, QPD platelets showed normal adherence to fibrin, but their adhesion was followed by accelerated fibrinolysis. The QPD is associated with "gain-of-function" abnormalities that increase the lysis of forming or preformed clots. These findings suggest accelerated fibrinolysis is an important contributor to QPD bleeding.

Activity of Nanobins Targeted to the Urokinase Plasminogen Activator System

NASA Astrophysics Data System (ADS)

Hankins, Patrick Leon

While innovations in nanotechnology have resulted in numerous medical advancements for the treatment of cancer, there remains an urgent unmet need for safe and efficient molecular platforms that facilitate the delivery of potent therapeutics to solid tumors. Nanoscale formulations help to overcome the poor bioavailability and systemic organ toxicity associated with many small molecule drugs. Of these nanoparticle drug delivery systems, the greatest clinical successes to date have employed simple nanoscale lipid bilayer assemblies which encase large payloads of chemotherapeutic. While the nanobin platform we have developed has seen initial success through the passive accumulation into tumors, actively targeting nanobins to tumor specific antigens has the potential to increase the therapeutic index of these nanoparticle drugs. We have identified the urokinase plasminogen activator (uPA) and its cell surface bound receptor (uPAR) as ideal targets for drug delivery due to their selective overexpression in metastatic cancers and their important role in tumor progression. From a panel of monoclonal antibodies targeted to uPA and uPAR, we have selected ATN291 and ATN658 as lead candidates for nanobin targeting based on their tumor cell binding and ability to be internalized by cells. A novel method of conjugating antibodies to liposomes was developed for our nanobin platform that preserves the high binding affinity and specificity of these antibodies. We evaluated these uPA- and uPAR-targeted nanobins in several xenograft tumor models and found that they were well-tolerated over a wide range of doses and demonstrated significantly increased antitumor efficacy over untargeted nanobins in multiple tumor types. Preliminary studies suggest that uPA-targeted nanobins are readily internalized by tumor cells, and we believe this is the mechanism for their increased antitumor effect. A method for radiolabeling nanobins with gallium-67 was developed, and preliminary SPECT-CT imaging studies showed the preferential accumulation of these nanobins in an orthotopic model of breast cancer. Due to their biocompatibility, robustness, and extensive history in the clinic, liposomes are an ideal drug delivery vehicle for the development of targeted therapies. The data presented in this thesis demonstrates the potential for active targeting to increase the therapeutic index of nanoscale drug delivery systems by increasing antitumor effect while simultaneously preventing drug uptake in peripheral tissue. In particular, targeting nanoparticles to the uPA system is a promising strategy for the treatment of many advanced, metastatic cancers.

A combination of desmopressin and docetaxel inhibit cell proliferation and invasion mediated by urokinase-type plasminogen activator (uPA) in human prostate cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sasaki, Hiroshi; Klotz, Laurence H.; Sugar, Linda M.

Background: This study was designed to assess the effectiveness of a combination treatment using both desmopressin and docetaxel in prostate cancer treatment. Desmopressin is a well-known synthetic analogue of the antidiuretic hormone vasopressin. It has recently been demonstrated to inhibit tumor progression and metastasis in in vivo models. Docetaxel is widely used for the treatment of castration resistant prostate cancer (CRPC) patients. However, durable responses have been uncommon to date. In this study, we investigated the anti-tumor effect of desmopressin in combination with docetaxel in vitro and in vivo. Methods: Two prostate cancer cells (PC3, LNCaP) were treated with different concentrations of desmopressinmore » alone, docetaxel alone, and a combination of desmopressin and docetaxel. Cell proliferation was determined by MTS assay. The anti-invasive and anti-migration potential of desmopressin and in combination with docetaxel were examined by wound healing assay, migration chamber assay, and matrigel invasion assay. Results: The combination of desmopressin and docetaxel resulted in a significant inhibition of PC3 and LNCaP cell proliferation (p < 0.01). Additionally, cell migration and invasion were also inhibited by the combination when compared to that of either treatment alone in PC3 cells (p < 0.01). The anti-tumor effect of this combination treatment was associated with down-regulation of both urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP-2 and MMP-9) in PC3 cells. Conclusions: We are the first to elucidate the anti-tumor and anti-metastatic potential of desmopressin in combination with docetaxel in a prostate cancer model via the uPA-MMP pathway. Our finding could potentially contribute to the therapeutic profile of desmopressin and enhance the efficacy of docetaxel based treatment for CRPC. - Highlights: • Desmopressin inhibits cell proliferation in prostate cancer cells. • The expression of cyclin A and CDK2 were reduced in the cells treated with desmopressin and docetaxel. • uPA-MMP pathway mediates the desmopressin-induced migration and invasion of PC3 cells. • Desmopressin in combination with docetaxel inhibits PC-3 cell growth in a xenograft model.« less

Soluble urokinase plasminogen activator receptor level is an independent predictor of the presence and severity of coronary artery disease and of future adverse events.

PubMed

Eapen, Danny J; Manocha, Pankaj; Ghasemzadeh, Nima; Ghasemzedah, Nima; Patel, Riyaz S; Al Kassem, Hatem; Hammadah, Muhammad; Veledar, Emir; Le, Ngoc-Anh; Pielak, Tomasz; Thorball, Christian W; Velegraki, Aristea; Kremastinos, Dimitrios T; Lerakis, Stamatios; Sperling, Laurence; Quyyumi, Arshed A

2014-10-23

Soluble urokinase plasminogen activator receptor (suPAR) is an emerging inflammatory and immune biomarker. Whether suPAR level predicts the presence and the severity of coronary artery disease (CAD), and of incident death and myocardial infarction (MI) in subjects with suspected CAD, is unknown. We measured plasma suPAR levels in 3367 subjects (67% with CAD) recruited in the Emory Cardiovascular Biobank and followed them for adverse cardiovascular (CV) outcomes of death and MI over a mean 2.1±1.1 years. Presence of angiographic CAD (≥50% stenosis in ≥1 coronary artery) and its severity were quantitated using the Gensini score. Cox's proportional hazard survival and discrimination analyses were performed with models adjusted for established CV risk factors and C-reactive protein levels. Elevated suPAR levels were independently associated with the presence of CAD (P<0.0001) and its severity (P<0.0001). A plasma suPAR level ≥3.5 ng/mL (cutoff by Youden's index) predicted future risk of MI (hazard ratio [HR]=3.2; P<0.0001), cardiac death (HR=2.62; P<0.0001), and the combined endpoint of death and MI (HR=1.9; P<0.0001), even after adjustment of covariates. The C-statistic for a model based on traditional risk factors was improved from 0.72 to 0.74 (P=0.008) with the addition of suPAR. Elevated levels of plasma suPAR are associated with the presence and severity of CAD and are independent predictors of death and MI in patients with suspected or known CAD. © 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Soluble Urokinase Plasminogen Activator Receptor Level Is an Independent Predictor of the Presence and Severity of Coronary Artery Disease and of Future Adverse Events

PubMed Central

Eapen, Danny J.; Manocha, Pankaj; Ghasemzedah, Nima; Patel, Riyaz S.; Al Kassem, Hatem; Hammadah, Muhammad; Veledar, Emir; Le, Ngoc‐Anh; Pielak, Tomasz; Thorball, Christian W.; Velegraki, Aristea; Kremastinos, Dimitrios T.; Lerakis, Stamatios; Sperling, Laurence; Quyyumi, Arshed A.

2014-01-01

Introduction Soluble urokinase plasminogen activator receptor (suPAR) is an emerging inflammatory and immune biomarker. Whether suPAR level predicts the presence and the severity of coronary artery disease (CAD), and of incident death and myocardial infarction (MI) in subjects with suspected CAD, is unknown. Methods and Results We measured plasma suPAR levels in 3367 subjects (67% with CAD) recruited in the Emory Cardiovascular Biobank and followed them for adverse cardiovascular (CV) outcomes of death and MI over a mean 2.1±1.1 years. Presence of angiographic CAD (≥50% stenosis in ≥1 coronary artery) and its severity were quantitated using the Gensini score. Cox's proportional hazard survival and discrimination analyses were performed with models adjusted for established CV risk factors and C‐reactive protein levels. Elevated suPAR levels were independently associated with the presence of CAD (P<0.0001) and its severity (P<0.0001). A plasma suPAR level ≥3.5 ng/mL (cutoff by Youden's index) predicted future risk of MI (hazard ratio [HR]=3.2; P<0.0001), cardiac death (HR=2.62; P<0.0001), and the combined endpoint of death and MI (HR=1.9; P<0.0001), even after adjustment of covariates. The C‐statistic for a model based on traditional risk factors was improved from 0.72 to 0.74 (P=0.008) with the addition of suPAR. Conclusion Elevated levels of plasma suPAR are associated with the presence and severity of CAD and are independent predictors of death and MI in patients with suspected or known CAD. PMID:25341887

A high-protein diet during hospitalization is associated with an accelerated decrease in soluble urokinase plasminogen activator receptor levels in acutely ill elderly medical patients with SIRS.

PubMed

Tavenier, Juliette; Haupt, Thomas H; Andersen, Aino L; Buhl, Sussi F; Langkilde, Anne; Andersen, Jens R; Jensen, Jens-Erik B; Pedersen, Mette M; Petersen, Janne; Andersen, Ove

2017-05-01

Acute illness and hospitalization in elderly individuals are often accompanied by the systemic inflammatory response syndrome (SIRS) and malnutrition, both associated with wasting and mortality. Nutritional support and resistance training were shown to increase muscle anabolism and reduce inflammation in healthy elderly. We hypothesized that nutritional support and resistance training would accelerate the resolution of inflammation in hospitalized elderly patients with SIRS. Acutely admitted patients aged >65 years with SIRS were randomized to an intervention consisting of a high-protein diet (1.7 g/kg per day) during hospitalization, and daily protein supplement (18.8 g) and 3 weekly resistance training sessions for 12 weeks after discharge (Intervention, n=14), or to standard-care (Control, n=15). Plasma levels of the inflammatory biomarkers soluble urokinase plasminogen activator receptor (suPAR), interleukin-6, C-reactive protein (CRP), and albumin were measured at admission, discharge, and 4 and 13 weeks after discharge. The Intervention group had an earlier decrease in suPAR levels than the Control group: -15.4% vs. +14.5%, P=.007 during hospitalization, and -2.4% vs. -28.6%, P=.007 between discharge and 4 weeks. There were no significant effects of the intervention on the other biomarkers. All biomarkers improved significantly between admission and 13 weeks, although with different kinetics (suPAR: -22%, interleukin-6: -86%, CRP: -89%, albumin: +11%). Nutritional support during hospitalization was associated with an accelerated decrease in suPAR levels, whereas the combined nutrition and resistance training intervention after discharge did not appear to affect the inflammatory state. Our results indicate that improved nutritional care during hospitalization may accelerate recovery in acutely ill elderly medical patients. Copyright © 2017 Elsevier Inc. All rights reserved.

mTORC2 activation is regulated by the urokinase receptor (uPAR) in bladder cancer.

PubMed

Hau, Andrew M; Leivo, Mariah Z; Gilder, Andrew S; Hu, Jing-Jing; Gonias, Steven L; Hansel, Donna E

2017-01-01

Mammalian target of rapamycin complex 2 (mTORC2) has been identified as a major regulator of bladder cancer cell migration and invasion. Upstream pathways that mediate mTORC2 activation remain poorly defined. Urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored membrane protein and known activator of cell-signaling. We identified increased uPAR expression in 94% of invasive human bladder cancers and in 54-71% of non-invasive bladder cancers, depending on grade. Normal urothelium was uPAR-immunonegative. Analysis of publicly available datasets identified uPAR gene amplification or mRNA upregulation in a subset of bladder cancer patients with reduced overall survival. Using biochemical approaches, we showed that uPAR activates mTORC2 in bladder cancer cells. Highly invasive bladder cancer cell lines, including T24, J82 and UM-UC-3 cells, showed increased uPAR mRNA expression and protein levels compared with the less aggressive cell lines, UROtsa and RT4. uPAR gene-silencing significantly reduced phosphorylation of Serine-473 in Akt, an mTORC2 target. uPAR gene-silencing also reduced bladder cancer cell migration and Matrigel invasion. S473 phosphorylation was observed by immunohistochemistry in human bladder cancers only when the tumors expressed high levels of uPAR. S473 phosphorylation was not controlled by uPAR in bladder cancer cell lines that are PTEN-negative; however, this result probably did not reflect altered mTORC2 regulation. Instead, PTEN deficiency de-repressed alternative kinases that phosphorylate S473. Our results suggest that uPAR and mTORC2 are components of a single cell-signaling pathway. Targeting uPAR or mTORC2 may be beneficial in patients with bladder cancer. Copyright © 2016. Published by Elsevier Inc.

In vitro regulation of pericellular proteolysis in prostatic tumor cells treated with bombesin.

PubMed

Festuccia, C; Guerra, F; D'Ascenzo, S; Giunciuglio, D; Albini, A; Bologna, M

1998-01-30

Bombesin is a potent inducer of signal trasduction pathways involved in the proliferation and invasion of androgen-insensitive prostatic tumor cells. This study examines the bombesin-mediated modulation of pericellular proteolysis, monitoring cell capability to migrate and invade basement membranes, using a chemo-invasion assay and analyzing protease production. The results suggest that bombesin could modulate the invasive potential of prostatic cell lines regulating secretion and cell-surface uptake of uPA and MMP-9 activation. In fact, in PC3 and DU145 cells but not in LNCaP cells, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) are induced by bombesin treatment. Bombesin also stimulates cell proliferation and this effect can be inhibited blocking uPA by antibodies and/or uPA inhibitor p-aminobenzamidine. Moreover, HMW-uPA induces cell proliferation in LNCaP cells, which do not produce uPA in the basal conditions, while PC3 and DU145 cell growth is supported by autocrine production of uPA. The increment of uPA activity on the external plasma membrane causes an increased pericellular plasmin activation. This effect is inhibited by antibodies against uPA and by p-aminobenzamidine. Similarly to EGF, bombesin stimulates secretion and activation of MMP-9 and TIMP-1 production. MMP-9 activation can be also obtained by HMW-uPA treatment, suggesting that plasma-membrane-bound uPA can start a proteolytic cascade involving MMP-9. Therefore, in in vitro assays, bombesin is able to modulate pericellular proteolysis and cell proliferation, differently distributing and activating proteolytic activities. This effect can be related to the "non-random" degradation of the extracellular matrix in which membrane uPA-uPAreceptor complexes could start bombesin-induced directional protein degradation during metastatic spread.

Co-ordinated spatial propagation of blood plasma clotting and fibrinolytic fronts

PubMed Central

Zhalyalov, Ansar S.; Panteleev, Mikhail A.; Gracheva, Marina A.; Ataullakhanov, Fazoil I.

2017-01-01

Fibrinolysis is a cascade of proteolytic reactions occurring in blood and soft tissues, which functions to disintegrate fibrin clots when they are no more needed. In order to elucidate its regulation in space and time, fibrinolysis was investigated using an in vitro reaction-diffusion experimental model of blood clot formation and dissolution. Clotting was activated by a surface with immobilized tissue factor in a thin layer of recalcified blood plasma supplemented with tissue plasminogen activator (TPA), urokinase plasminogen activator or streptokinase. Formation and dissolution of fibrin clot was monitored by videomicroscopy. Computer systems biology model of clot formation and lysis was developed for data analysis and experimental planning. Fibrin clot front propagated in space from tissue factor, followed by a front of clot dissolution propagating from the same source. Velocity of lysis front propagation linearly depended on the velocity clotting front propagation (correlation r2 = 0.91). Computer model revealed that fibrin formation was indeed the rate-limiting step in the fibrinolysis front propagation. The phenomenon of two fronts which switched the state of blood plasma from liquid to solid and then back to liquid did not depend on the fibrinolysis activator. Interestingly, TPA at high concentrations began to increase lysis onset time and to decrease lysis propagation velocity, presumably due to plasminogen depletion. Spatially non-uniform lysis occurred simultaneously with clot formation and detached the clot from the procoagulant surface. These patterns of spatial fibrinolysis provide insights into its regulation and might explain clinical phenomena associated with thrombolytic therapy. PMID:28686711

A novel embryo culture media supplement that improves pregnancy rates in mice.

PubMed

Highet, A R; Bianco-Miotto, T; Pringle, K G; Peura, A; Bent, S; Zhang, J; Nottle, M B; Thompson, J G; Roberts, C T

2017-03-01

The preimplantation embryo in vivo is exposed to numerous growth factors in the female reproductive tract, which are not recapitulated in embryo culture media in vitro The IGF2 and plasminogen activator systems facilitate blastocyst development. We hypothesized that the addition of IGF2 in combination with urokinase plasminogen activator (uPA) and plasminogen could improve rates of blastocyst hatching and implantation in mice. B6BcF1 and CBAB6F2 mouse embryos were divided into one of four supplemented culture media treatment groups: (1) control (media only); (2) 12.5 nM IGF2; (3) 10 µg/mL uPA and 5 µg/mL plasminogen; or (4) a combination of IGF2, uPA and plasminogen treatments. Embryo development to blastocyst stage and hatching were assessed before transfer to pseudopregnant recipient females and implantation, pregnancy rates and postnatal growth were assessed. After 90.5 h of culture, IGF2 + U + P treatment increased the percentage of B6BcF1 embryos that were hatching/hatched and percentage developing to blastocyst stage compared with controls (P < 0.02). Following B6BcF1 embryo transfer, IGF2 + U + P treatment increased implantation sites at day 8 of pregnancy compared with controls (P < 0.05). Replication in the CBAB6F2 mouse strain showed significant improvements in pregnancy rates at days 8 and 18 but not in blastocyst development. No adverse effects were seen on gestational age, litter size or birthweight, or the reproductive capacity of offspring of IGF2 + U + P treated embryos. For embryos susceptible to detrimental effects of in vitro culture, IGF2, uPA and plasminogen supplementation of culture media can improve pregnancy success, but the effect of treatment is dependent on the mouse strain. © 2017 Society for Reproduction and Fertility.

Inhibitory Effects of North American Wild Rice on Monocyte Adhesion and Inflammatory Modulators in Low-Density Lipoprotein Receptor-Knockout Mice.

PubMed

Moghadasian, Mohammed H; Zhao, Ruozhi; Ghazawwi, Nora; Le, Khuong; Apea-Bah, Franklin B; Beta, Trust; Shen, Garry X

2017-10-18

The present study examined the effects of wild rice on monocyte adhesion, inflammatory and fibrinolytic mediators in low-density lipoprotein receptor-knockout (LDLr-KO) mice. Male LDLr-KO mice received a cholesterol (0.06%, w/w)-supplemented diet with or without white or wild rice (60%, w/w) for 20 weeks. White rice significantly increased monocyte adhesion and abundances of monocyte chemoattractant protein-1, tissue necrosis factor-α, intracellular cell adhesion molecule-1, plasminogen activator inhibitor-1, urokinase plasminogen activator (uPA), and uPA receptor in aortae and hearts of LDLr-KO mice compared to the control diet. Wild rice inhibited monocyte adhesion to the aorta, atherosclerosis, and abundances of the inflammatory and fibrinolytic regulators in the cardiovascular tissue of LDLr-KO mice compared to white rice. White or wild rice did not significantly alter the levels of cholesterol, triglycerides, or antioxidant enzymes in plasma. The anti-atherosclerotic effect of wild rice may result from its inhibition on monocyte adhesion and inflammatory modulators in LDLr-KO mice.

[Cell-derived microparticles unveil their fibrinolytic and proteolytic function].

PubMed

Doeuvre, Loïc; Angles-Cano, Eduardo

2009-01-01

Cell-derived microparticles (MP) are membrane microvesicles, 0.1-1 microm in size, shed by cells following activation or during apoptosis in a variety of pathological conditions. MPs released by blood cells or by vascular endothelial cells display molecular signatures that allow their identification and functional characterization. In addition, they provide tissue factor (TF) and a procoagulant phospholipid surface. Therefore, at present, the most strongly established applied research on MPs is their procoagulant activity as a determinant of thrombotic risk in various clinical conditions. Previous studies have indicated that MPs derived from malignant cells express matrix metalloproteinases, urokinase and its receptor (uPA/uPAR) that, in the presence of plasminogen, may act in concert to degrade extracellular matrix proteins. Recently, it was shown that MPs from TNFa-stimulated endothelial cells served as a surface for interaction with plasminogen and its conversion into plasmin by the uPA/uPAR system expressed at their surface. This capacity of MPs to promote plasmin generation confers them a new profibrinolytic and proteolytic function that may be of relevance in fibrinolysis, cell migration, angiogenesis, dissemination of malignant cells, cell detachment and apoptosis.

Active α-macroglobulin is a reservoir for urokinase after fibrinolytic therapy in rabbits with tetracycline-induced pleural injury and in human pleural fluids

PubMed Central

Florova, Galina; Azghani, Ali; Karandashova, Sophia; Kurdowska, Anna K.; Idell, Steven

2013-01-01

Intrapleural processing of prourokinase (scuPA) in tetracycline (TCN)-induced pleural injury in rabbits was evaluated to better understand the mechanisms governing successful scuPA-based intrapleural fibrinolytic therapy (IPFT), capable of clearing pleural adhesions in this model. Pleural fluid (PF) was withdrawn 0–80 min and 24 h after IPFT with scuPA (0–0.5 mg/kg), and activities of free urokinase (uPA), plasminogen activator inhibitor-1 (PAI-1), and uPA complexed with α-macroglobulin (αM) were assessed. Similar analyses were performed using PFs from patients with empyema, parapneumonic, and malignant pleural effusions. The peak of uPA activity (5–40 min) reciprocally correlated with the dose of intrapleural scuPA. Endogenous active PAI-1 (10–20 nM) decreased the rate of intrapleural scuPA activation. The slow step of intrapleural inactivation of free uPA (t1/2β = 40 ± 10 min) was dose independent and 6.7-fold slower than in blood. Up to 260 ± 70 nM of αM/uPA formed in vivo [second order association rate (kass) = 580 ± 60 M−1·s−1]. αM/uPA and products of its degradation contributed to durable intrapleural plasminogen activation up to 24 h after IPFT. Active PAI-1, active α2M, and α2M/uPA found in empyema, pneumonia, and malignant PFs demonstrate the capacity to support similar mechanisms in humans. Intrapleural scuPA processing differs from that in the bloodstream and includes 1) dose-dependent control of scuPA activation by endogenous active PAI-1; 2) two-step inactivation of free uPA with simultaneous formation of αM/uPA; and 3) slow intrapleural degradation of αM/uPA releasing active free uPA. This mechanism offers potential clinically relevant advantages that may enhance the bioavailability of intrapleural scuPA and may mitigate the risk of bleeding complications. PMID:23997178

Could soluble urokinase plasminogen receptor (suPAR) be used as a diagnostic biomarker for ventilator-associated pneumonia?

PubMed

Sunnetcioglu, Aysel; Sunnetcioglu, Mahmut; Adıyaman, Fırat; Binici, Irfan; Soyoral, Lokman

2017-11-01

Soluble urokinase plasminogen activator receptor (suPAR) is a biomarker that is increasingly used for evaluation of systemic inflammation. This study was performed to investigate whether suPAR may possess a diagnostic value in patients with ventilator-associated pneumonia (VAP). This clinical study was performed in the anesthesia intensive care units (ICUs) of our university. In addition to descriptive data, WBC, serum levels of C-reactive protein (CRP) and suPAR prior to and after development of VAP were noted and compared in 31 patients (22 men, 9 women) diagnosed with VAP (Study Group) and 19 patients without VAP (Control Group) in ICU (14 men, 5 women). The suPAR (P = 0.023), CRP (P = 0.037), WBCs (P = 0.024) in patients with VAP were significantly higher than patients without VAP. There was no remarkable difference in terms of WBCs (P = 0.052) and suPAR levels (P = 0.616) between groups on the first day of connection to mechanical ventilator. The suPAR and CRP levels in patients with VAP were significantly higher than prior to development of VAP (P = 0.001 for both). Area under curve value after diagnosis of pneumonia was found 0.248 (P = 0.002). To conclude, our results suggest that suPAR can be a useful diagnostic biomarker in patients with VAP. However, clinical trials on larger series are warranted to explore the clinical significance more accurately. © 2016 John Wiley & Sons Ltd.

Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries

PubMed Central

Harris, Jennifer L.; Backes, Bradley J.; Leonetti, Francesco; Mahrus, Sami; Ellman, Jonathan A.; Craik, Charles S.

2000-01-01

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors. PMID:10869434

Use of Biomarkers to Guide Decisions on Adjuvant Systemic Therapy for Women With Early-Stage Invasive Breast Cancer: American Society of Clinical Oncology Clinical Practice Guideline.

PubMed

Harris, Lyndsay N; Ismaila, Nofisat; McShane, Lisa M; Andre, Fabrice; Collyar, Deborah E; Gonzalez-Angulo, Ana M; Hammond, Elizabeth H; Kuderer, Nicole M; Liu, Minetta C; Mennel, Robert G; Van Poznak, Catherine; Bast, Robert C; Hayes, Daniel F

2016-04-01

To provide recommendations on appropriate use of breast tumor biomarker assay results to guide decisions on adjuvant systemic therapy for women with early-stage invasive breast cancer. A literature search and prospectively defined study selection sought systematic reviews, meta-analyses, randomized controlled trials, prospective-retrospective studies, and prospective comparative observational studies published from 2006 through 2014. Outcomes of interest included overall survival and disease-free or recurrence-free survival. Expert panel members used informal consensus to develop evidence-based guideline recommendations. The literature search identified 50 relevant studies. One randomized clinical trial and 18 prospective-retrospective studies were found to have evaluated the clinical utility, as defined by the guideline, of specific biomarkers for guiding decisions on the need for adjuvant systemic therapy. No studies that met guideline criteria for clinical utility were found to guide choice of specific treatments or regimens. In addition to estrogen and progesterone receptors and human epidermal growth factor receptor 2, the panel found sufficient evidence of clinical utility for the biomarker assays Oncotype DX, EndoPredict, PAM50, Breast Cancer Index, and urokinase plasminogen activator and plasminogen activator inhibitor type 1 in specific subgroups of breast cancer. No biomarker except for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 was found to guide choices of specific treatment regimens. Treatment decisions should also consider disease stage, comorbidities, and patient preferences. © 2016 by American Society of Clinical Oncology.

Use of Biomarkers to Guide Decisions on Adjuvant Systemic Therapy for Women With Early-Stage Invasive Breast Cancer: American Society of Clinical Oncology Clinical Practice Guideline

PubMed Central

Harris, Lyndsay N.; McShane, Lisa M.; Andre, Fabrice; Collyar, Deborah E.; Gonzalez-Angulo, Ana M.; Hammond, Elizabeth H.; Kuderer, Nicole M.; Liu, Minetta C.; Mennel, Robert G.; Van Poznak, Catherine; Bast, Robert C.; Hayes, Daniel F.

2016-01-01

Purpose To provide recommendations on appropriate use of breast tumor biomarker assay results to guide decisions on adjuvant systemic therapy for women with early-stage invasive breast cancer. Methods A literature search and prospectively defined study selection sought systematic reviews, meta-analyses, randomized controlled trials, prospective-retrospective studies, and prospective comparative observational studies published from 2006 through 2014. Outcomes of interest included overall survival and disease-free or recurrence-free survival. Expert panel members used informal consensus to develop evidence-based guideline recommendations. Results The literature search identified 50 relevant studies. One randomized clinical trial and 18 prospective-retrospective studies were found to have evaluated the clinical utility, as defined by the guideline, of specific biomarkers for guiding decisions on the need for adjuvant systemic therapy. No studies that met guideline criteria for clinical utility were found to guide choice of specific treatments or regimens. Recommendations In addition to estrogen and progesterone receptors and human epidermal growth factor receptor 2, the panel found sufficient evidence of clinical utility for the biomarker assays Oncotype DX, EndoPredict, PAM50, Breast Cancer Index, and urokinase plasminogen activator and plasminogen activator inhibitor type 1 in specific subgroups of breast cancer. No biomarker except for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 was found to guide choices of specific treatment regimens. Treatment decisions should also consider disease stage, comorbidities, and patient preferences. PMID:26858339

Plasminogen activator inhibitor-2 in patients with monocytic leukemia.

PubMed

Scherrer, A; Kruithof, E K; Grob, J P

1991-06-01

Plasma and tumor cells from 103 patients with leukemia or lymphoma at initial presentation were investigated for the presence of plasminogen activator inhibitor-2 (PAI-2) antigen, a potent inhibitor of urokinase. PAI-2 was detected in plasma and leukemic cells of the 21 patients with leukemia having a monocytic component [acute myelomonocytic (M4), acute monoblastic (M5), and chronic myelomonocytic leukemias], and in the three patients with acute undifferentiated myeloblastic leukemia (M0). In contrast, this serine protease inhibitor was undetectable in 79 patients with other subtypes of acute myeloid leukemia or other hematological malignancies. Serial serum PAI-2 determinations in 16 patients with acute leukemia at presentation, during therapy, remission, and relapse revealed that in the five patients with M4-M5, elevated PAI-2 levels rapidly normalized under therapy and during remission, but increased again in the patients with a relapse associated with an M4-M5 phenotype. Thus, PAI-2 seems to be a marker highly specific for the active stages of monocytic leukemia, i.e. presentation and relapse. The presence of PAI-2 in the plasma and cells of patients with M0 may give a clue to a monocytic origin of these cells.

Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination.

PubMed

Nakatsuka, Erika; Sawada, Kenjiro; Nakamura, Koji; Yoshimura, Akihito; Kinose, Yasuto; Kodama, Michiko; Hashimoto, Kae; Mabuchi, Seiji; Makino, Hiroshi; Morii, Eiichi; Yamaguchi, Yoichi; Yanase, Takeshi; Itai, Akiko; Morishige, Ken-Ichirou; Kimura, Tadashi

2017-10-27

In the present study, the therapeutic potential of targeting plasminogen activator inhibitor-1 (PAI-1) in ovarian cancer was tested. Tissues samples from 154 cases of ovarian carcinoma were immunostained with anti-PAI-1 antibody, and the prognostic value was analyzed. Among the samples, 67% (104/154) showed strong PAI-1 expression; this was significantly associated with poor prognosis (progression-free survival: 20 vs. 31 months, P = 0.0033). In particular, among patients with stage II-IV serous adenocarcinoma, PAI-1 expression was an independent prognostic factor. The effect of a novel PAI-1 inhibitor, IMD-4482, on ovarian cancer cell lines was assessed and its therapeutic potential was examined using a xenograft mouse model of ovarian cancer. IMD-4482 inhibited in vitro cell adhesion to vitronectin in PAI-1-positive ovarian cancer cells, followed by the inhibition of extracellular signal-regulated kinase and focal adhesion kinase phosphorylation through dissociation of the PAI-urokinase receptor complex from integrin αVβ3. IMD-4482 caused G0/G1 cell arrest and inhibited the proliferation of PAI-1-positive ovarian cancer cells. In the xenograft model, IMD-4482 significantly inhibited peritoneal dissemination with the reduction of PAI-1 expression and the inhibition of focal adhesion kinase phosphorylation. Collectively, the functional inhibition of PAI-1 significantly inhibited ovarian cancer progression, and targeting PAI-1 may be a potential therapeutic strategy in ovarian cancer.

Plasminogen activator inhibitor-1 is an independent prognostic factor of ovarian cancer and IMD-4482, a novel plasminogen activator inhibitor-1 inhibitor, inhibits ovarian cancer peritoneal dissemination

PubMed Central

Nakatsuka, Erika; Sawada, Kenjiro; Nakamura, Koji; Yoshimura, Akihito; Kinose, Yasuto; Kodama, Michiko; Hashimoto, Kae; Mabuchi, Seiji; Makino, Hiroshi; Morii, Eiichi; Yamaguchi, Yoichi; Yanase, Takeshi; Itai, Akiko; Morishige, Ken-ichirou; Kimura, Tadashi

2017-01-01

In the present study, the therapeutic potential of targeting plasminogen activator inhibitor-1 (PAI-1) in ovarian cancer was tested. Tissues samples from 154 cases of ovarian carcinoma were immunostained with anti-PAI-1 antibody, and the prognostic value was analyzed. Among the samples, 67% (104/154) showed strong PAI-1 expression; this was significantly associated with poor prognosis (progression-free survival: 20 vs. 31 months, P = 0.0033). In particular, among patients with stage II-IV serous adenocarcinoma, PAI-1 expression was an independent prognostic factor. The effect of a novel PAI-1 inhibitor, IMD-4482, on ovarian cancer cell lines was assessed and its therapeutic potential was examined using a xenograft mouse model of ovarian cancer. IMD-4482 inhibited in vitro cell adhesion to vitronectin in PAI-1-positive ovarian cancer cells, followed by the inhibition of extracellular signal-regulated kinase and focal adhesion kinase phosphorylation through dissociation of the PAI-urokinase receptor complex from integrin αVβ3. IMD-4482 caused G0/G1 cell arrest and inhibited the proliferation of PAI-1-positive ovarian cancer cells. In the xenograft model, IMD-4482 significantly inhibited peritoneal dissemination with the reduction of PAI-1 expression and the inhibition of focal adhesion kinase phosphorylation. Collectively, the functional inhibition of PAI-1 significantly inhibited ovarian cancer progression, and targeting PAI-1 may be a potential therapeutic strategy in ovarian cancer. PMID:29163796

Plasminogen Activator Inhibitor-1 Deficiency Augments Visceral Mesothelial Organization, Intrapleural Coagulation, and Lung Restriction in Mice with Carbon Black/Bleomycin–Induced Pleural Injury

PubMed Central

Jeffers, Ann; Alvarez, Alexia; Owens, Shuzi; Koenig, Kathleen; Quaid, Brandon; Komissarov, Andrey A.; Florova, Galina; Kothari, Hema; Pendurthi, Usha; Mohan Rao, L. Vijaya; Idell, Steven

2014-01-01

Local derangements of fibrin turnover and plasminogen activator inhibitor (PAI)-1 have been implicated in the pathogenesis of pleural injury. However, their role in the control of pleural organization has been unclear. We found that a C57Bl/6j mouse model of carbon black/bleomycin (CBB) injury demonstrates pleural organization resulting in pleural rind formation (14 d). In transgenic mice overexpressing human PAI-1, intrapleural fibrin deposition was increased, but visceral pleural thickness, lung volumes, and compliance were comparable to wild type. CBB injury in PAI-1−/− mice significantly increased visceral pleural thickness (P < 0.001), elastance (P < 0.05), and total lung resistance (P < 0.05), while decreasing lung compliance (P < 0.01) and lung volumes (P < 0.05). Collagen, α-smooth muscle actin, and tissue factor were increased in the thickened visceral pleura of PAI-1−/− mice. Colocalization of α-smooth muscle actin and calretinin within pleural mesothelial cells was increased in CBB-injured PAI-1−/− mice. Thrombin, factor Xa, plasmin, and urokinase induced mesothelial–mesenchymal transition, tissue factor expression, and activity in primary human pleural mesothelial cells. In PAI-1−/− mice, D-dimer and thrombin–antithrombin complex concentrations were increased in pleural lavage fluids. The results demonstrate that PAI-1 regulates CBB-induced pleural injury severity via unrestricted fibrinolysis and cross-talk with coagulation proteases. Whereas overexpression of PAI-1 augments intrapleural fibrin deposition, PAI-1 deficiency promotes profibrogenic alterations of the mesothelium that exacerbate pleural organization and lung restriction. PMID:24024554

Up-Regulation of PAI-1 and Down-Regulation of uPA Are Involved in Suppression of Invasiveness and Motility of Hepatocellular Carcinoma Cells by a Natural Compound Berberine.

PubMed

Wang, Xuanbin; Wang, Ning; Li, Hongliang; Liu, Ming; Cao, Fengjun; Yu, Xianjun; Zhang, Jingxuan; Tan, Yan; Xiang, Longchao; Feng, Yibin

2016-04-16

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death and its prognosis remains poor due to the high risk of tumor recurrence and metastasis. Berberine (BBR) is a natural compound derived from some medicinal plants, and accumulating evidence has shown its potent anti-tumor activity with diverse action on tumor cells, including inducing cancer cell death and blocking cell cycle and migration. Molecular targets of berberine involved in its inhibitory effect on the invasiveness remains not yet clear. In this study, we identified that berberine exhibits a potent inhibition on the invasion and migration of HCC cells. This was accompanied by a dose-dependent down-regulation of expression of Cyclooxygenase-2 (COX-2), nuclear factor kappa B (NF-κB), urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9 in berberine-treated HCC cells. Furthermore, berberine inactivated p38 and Erk1/2 signaling pathway in HCC cells. Primarily, this may be attributed to the up-regulation of plasminogen activator inhibitor-1 (PAI-1), a tumor suppressor that can antagonize uPA receptor and down-regulation of uPA. Blockade of uPA receptor-associated pathways leads to reduced invasiveness and motility of berberine-treated HCC cells. In conclusion, our findings identified for the first time that inactivation of uPA receptor by up-regulation of PAI-1 and down-regulation of uPA is involved in the inhibitory effect of berberine on HCC cell invasion and migration.

Hyperglycemia-induced mouse trophoblast spreading is mediated by reactive oxygen species.

PubMed

Sánchez-Santos, Alejandra; Martínez-Hernández, María G; Contreras-Ramos, Alejandra; Ortega-Camarillo, Clara; Baiza-Gutman, Luis A

2018-04-01

During embryo implantation, the outer layer of the blastocyst interacts with the endometrium giving rise to the development of the trophoblast cell lineage. The cells in this lineage participate in the penetration of endometrium due to their motility and invasive properties. The mechanisms that regulate the differentiation and invasive ability of these cells are essential for the establishment and maintenance of an efficient exchange between maternal and fetal tissues during pregnancy. In this context, hyperglycemia can induce oxidative stress causing alterations in the placenta. This study evaluated the role of reactive oxygen species (ROS) in the actions of high glucose concentration (HG) on trophoblast spreading and the expression of extracellular proteases in cultured mouse conceptuses. Blastocysts from gestational day 4 (GD4) were cultured until GD7 in HAM-F10 medium and further treated for 48 hr with HG (25 mM glucose) from GD7 to GD9. This treatment induced larger trophoblast outgrowths and increased ROS concentration, which was associated with increased expression levels of urokinase-type plasminogen activator (PLAU), plasminogen activator inhibitor 1 (PAI-1), and matrix metalloproteinase 9 (MMP-9). These effects were prevented by treatment with the non-specific antioxidant N-acetylcysteine (NAC) or apocynin, an inhibitor of NADPH oxidase. Our data suggest that the HG-induced trophoblast spreading and the expression of PLAU, PAI-1, and MMP-9 were mediated by the production of ROS via NADPH oxidase activity. Our results shed light on placental alterations in gestational diabetes mellitus. © 2018 Wiley Periodicals, Inc.

Annexin II-Dependent Mechanism of Breast Cancer Progression

DTIC Science & Technology

2010-11-01

of the effects of tranexamic acid and urokinase on metastasis of Lewis lung carcinoma. Br. J. Cancer 46, 428–435. Tarui, T., et al., 2002. Plasmin...Lysine–Sepharose and ε- aminocaproic acid (ε-ACA)were procured from Sigma (St. Louis, MO). Chromatography and electrophoretic reagents were procured from...μl) with medium containing 10% fetal bovine serum (FBS). Test reagents (plasminogen, plasmin, angiostatin, ε-aminocaproic acid , or antibo- dies) were

Cleavage of the urokinase receptor (uPAR) on oral cancer cells: regulation by transforming growth factor - β1 (TGF-β1) and potential effects on migration and invasion.

PubMed

Magnussen, Synnove Norvoll; Hadler-Olsen, Elin; Costea, Daniela Elena; Berg, Eli; Jacobsen, Cristiane Cavalcanti; Mortensen, Bente; Salo, Tuula; Martinez-Zubiaurre, Inigo; Winberg, Jan-Olof; Uhlin-Hansen, Lars; Svineng, Gunbjorg

2017-05-19

Urokinase plasminogen activator (uPA) receptor (uPAR) is up-regulated at the invasive tumour front of human oral squamous cell carcinoma (OSCC), indicating a role for uPAR in tumour progression. We previously observed elevated expression of uPAR at the tumour-stroma interface in a mouse model for OSCC, which was associated with increased proteolytic activity. The tumour microenvironment regulated uPAR expression, as well as its glycosylation and cleavage. Both full-length- and cleaved uPAR (uPAR (II-III)) are involved in highly regulated processes such as cell signalling, proliferation, migration, stem cell mobilization and invasion. The aim of the current study was to analyse tumour associated factors and their effect on uPAR cleavage, and the potential implications for cell proliferation, migration and invasion. Mouse uPAR was stably overexpressed in the mouse OSCC cell line AT84. The ratio of full-length versus cleaved uPAR as analysed by Western blotting and its regulation was assessed by addition of different protease inhibitors and transforming growth factor - β1 (TGF-β1). The role of uPAR cleavage in cell proliferation and migration was analysed using real-time cell analysis and invasion was assessed using the myoma invasion model. We found that when uPAR was overexpressed a proportion of the receptor was cleaved, thus the cells presented both full-length uPAR and uPAR (II-III). Cleavage was mainly performed by serine proteases and urokinase plasminogen activator (uPA) in particular. When the OSCC cells were stimulated with TGF-β1, the production of the uPA inhibitor PAI-1 was increased, resulting in a reduction of uPAR cleavage. By inhibiting cleavage of uPAR, cell migration was reduced, and by inhibiting uPA activity, invasion was reduced. We could also show that medium containing soluble uPAR (suPAR), and cleaved soluble uPAR (suPAR (II-III)), induced migration in OSCC cells with low endogenous levels of uPAR. These results show that soluble factors in the tumour microenvironment, such as TGF-β1, PAI-1 and uPA, can influence the ratio of full length and uPAR (II-III) and thereby potentially effect cell migration and invasion. Resolving how uPAR cleavage is controlled is therefore vital for understanding how OSCC progresses and potentially provides new targets for therapy.

Kidney cell electrophoresis in space flight: Rationale, methods, results and flow cytometry applications

NASA Technical Reports Server (NTRS)

Todd, P.; Morrison, Dennis R.; Barlow, Grant H.; Lewis, Marian L.; Lanham, J. W.; Cleveland, C.; Williams, K.; Kunze, M. E.; Goolsby, C. L.

1988-01-01

Cultures of human embryonic kidney cells consistently contain an electrophoretically separable subpopulation of cells that produce high levels of urokinase and have an electrophoretic mobility about 85 percent as high as that of the most mobile human embryonic kidney cells. This subpopulation is rich in large epithelioid cells that have relatively little internal structure. When resolution and throughput are adequate, free fluid electrophoresis can be used to isolate a broad band of low mobility cells which also produces high levels of plasminogen activators (PAs). In the course of performing this, it was discovered that all electrophoretic subpopulations of cultured human embryonic kidney cells produce some PAs and that separate subpopulations produce high quantities of different types of PA's. This information and the development of sensitive assays for this project have provided new insights into cell secretion mechanisms related to fibrinolysis. These advances would probably not have been made without the NASA program to explore fundamental questions of free fluid electrophoresis in space.

A peptide inhibitor of the urokinase/urokinase receptor system inhibits alteration of the blood-retinal barrier in diabetes.

PubMed

Navaratna, Deepti; Menicucci, Gina; Maestas, Joann; Srinivasan, Ramprasad; McGuire, Paul; Das, Arup

2008-09-01

One of the major complications of diabetes is the alteration of the blood-retinal barrier, leading to retinal edema and consequent vision loss. The aim of this study was to evaluate the role of the urokinase plasminogen activator (uPA)/uPA receptor (uPAR) system in the regulation of retinal vascular permeability. Biochemical, molecular, and histological techniques were used to examine the role of uPA and uPAR in the regulation of retinal vascular permeability in diabetic rats and cultured retinal endothelial cells. The increased retinal vascular permeability in diabetic rats was associated with a decrease in vascular endothelial (VE) -cadherin expression in retinal vessels. Treatment with the uPA/uPAR-inhibiting peptide (A6) was shown to reduce diabetes-induced permeability and the loss of VE-cadherin. The increased permeability of cultured cells in response to advanced glycation end products (AGEs) was significantly inhibited with A6. Treatment of endothelial cells with specific matrix metalloproteinases or AGEs resulted in loss of VE-cadherin from the cell surface, which could be inhibited by A6. uPA/uPAR physically interacts with AGEs/receptor for advanced glycation end products on the cell surface and regulates its activity. uPA and its receptor uPAR play important roles in the alteration of the blood-retinal barrier through proteolytic degradation of VE-cadherin. The ability of A6 to block retinal vascular permeability in diabetes suggests a potential therapeutic approach for the treatment of diabetic macular edema.

Properties of electrophoretic fractions of human embryonic kidney cells separated on space shuttle flight STS-8

NASA Technical Reports Server (NTRS)

Morrison, D. R.; Lewis, M. L.; Barlow, G. H.; Todd, P. W.; Kunze, M. E.; Sarnoff, B. E.; Li, Z. K.

1985-01-01

Suspensions of cultured primary human embryonic kidney cells were subjected to continuous flow electrophoresis on Space Shuttle flight STS-8. The objectives of the experiments were to obtain electrophoretically separated fractions of the original cell populations and to test these fractions for the amount and kind of urokinase (a kidney plasminogen activator that is used medically for digesting blood clots), the morphologies of cells in the individual fractions, and their cellular electrophoretic mobilities after separation and subsequent proliferation. Individual fractions were successfully cultured after return from orbit, and they were found to differ substantially from one another and from the starting sample with respect to all of these properties.

Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernhart, Eva; Damm, Sabine; Wintersperger, Andrea

Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which, despite combined modality treatment, reoccurs and is invariably fatal for affected patients. Recently, a member of the serine/threonine protein kinase D (PRKD) family, PRKD2, was shown to be a potent mediator of glioblastoma growth. Here we studied the role of PRKD2 in U87MG glioblastoma cell migration and invasion in response to sphingosine-1-phosphate (S1P), an activator of PRKD2 and a GBM mitogen. Time-lapse microscopy demonstrated that random cell migration was significantly diminished in response to PRKD2 silencing. The pharmacological PRKD family inhibitor CRT0066101 decreased chemotactic migration and invasion across uncoatedmore » or matrigel-coated Transwell inserts. Silencing of PRKD2 attenuated migration and invasion of U87MG cells even more effectively. In terms of downstream signaling, CRT0066101 prevented PRKD2 autophosphorylation and inhibited p44/42 MAPK and to a smaller extent p54/46 JNK and p38 MAPK activation. PRKD2 silencing impaired activation of p44/42 MAPK and p54/46 JNK, downregulated nuclear c-Jun protein levels and decreased c-Jun{sup S73} phosphorylation without affecting the NFκB pathway. Finally, qPCR array analyses revealed that silencing of PRKD2 downregulates mRNA levels of integrin alpha-2 and -4 (ITGA2 and -4), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and matrix metallopeptidase 1 (MMP1). Findings of the present study identify PRKD2 as a potential target to interfere with glioblastoma cell migration and invasion, two major determinants contributing to recurrence of glioblastoma after multimodality treatment. Highlights: • Sphingosine-1-phosphate induces glioma cell migration and invasion. • Part of the effects is mediated by protein kinase D2 (PRKD2) activation. • Inactivation of PRKD2 attenuates glioblastoma cell migration and invasion. • Both, RNAi and pharmacological inhibition of PRKD2 inhibits MAPK signaling. • PRKD2 regulates transcription of gene products implicated in migration and invasion.« less

N2 non-thermal atmospheric pressure plasma promotes wound healing in vitro and in vivo: Potential modulation of adhesion molecules and matrix metalloproteinase-9.

PubMed

Kang, Sung Un; Choi, Jae Won; Chang, Jae Won; Kim, Kang Il; Kim, Yeon Soo; Park, Ju Kyeong; Kim, Yang Eun; Lee, Yun Sang; Yang, Sang Sik; Kim, Chul-Ho

2017-02-01

Advances in physics and biology have made it possible to apply non-thermal atmospheric pressure plasma (NTP) in the biomedical field. Although accumulating evidence suggests that NTP has various medicinal effects, such as facilitating skin wound healing on exposed tissue while minimizing undesirable tissue damage, the underlying molecular mechanisms are not fully understood. In this study, NTP generated from N 2 optimized wound healing in the scratch wound healing assay. In addition, matrix metalloproteinase (MMP)-9 expression and enzyme activity increased and the urokinase-type plasminogen activator (uPA) system was activated after NTP treatment. We also showed that NTP treatment increased Slug and TCF8/ZEB1 expression and decreased that of E-cadherin, suggesting induction of the epithelial-to-mesenchymal transition (EMT). The effect of N 2 NTP was verified on rat wound model. Taken together, these results suggest that N 2 NTP promotes wound healing by inducing the EMT and activating the MMP-9/uPA system. These findings show the therapeutic potential of NTP for skin wound healing. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor: a study based on biosensor technology.

PubMed

List, K; Høyer-Hansen, G; Rønne, E; Danø, K; Behrendt, N

1999-01-01

Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interference with conformational properties of the receptor critical for ligand binding. This distinction is central when employing the antibodies as tools in the elucidation of the structure-function relationship of the protein in question. We have studied the effect of monoclonal antibodies against the urokinase plasminogen activator receptor (uPAR), a protein located on the surface of various types of malignant and normal cells which is involved in the direction of proteolytic degradation reactions in the extracellular matrix. We show that surface plasmon resonance/biomolecular interaction analysis (BIA) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former antibody efficiently blocked the receptor against subsequent ligand binding but was unable to promote the dissociation of a preformed receptor-ligand complex. The latter antibody was capable of binding the preformed complex, forming a transient trimolecular assembly, and promoting the dissociation of the uPA/uPAR complex. The continuous recording of binding and dissociation, obtained in BIA, is central in characterizing these phenomena. The identification of a non-competitive inhibitory mechanism against this receptor reveals the presence of a determinant which influences the binding properties of a remote site in the molecular structure and which could be an important target for a putative synthetic antagonist.

Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein-Protein Interaction.

PubMed

Liu, Degang; Xu, David; Liu, Min; Knabe, William Eric; Yuan, Cai; Zhou, Donghui; Huang, Mingdong; Meroueh, Samy O

2017-03-28

Protein-protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein-protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein-protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π-cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π-cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein-protein interactions.

Interactions of surface-displayed glycolytic enzymes of Mycoplasma pneumoniae with components of the human extracellular matrix.

PubMed

Gründel, Anne; Jacobs, Enno; Dumke, Roger

2016-12-01

Mycoplasma pneumoniae is a major cause of community-acquired respiratory infections worldwide. Due to the strongly reduced genome, the number of virulence factors expressed by this cell wall-less pathogen is limited. To further understand the processes during host colonization, we investigated the interactions of the previously confirmed surface-located glycolytic enzymes of M. pneumoniae (pyruvate dehydrogenase A-C [PdhA-C], glyceraldehyde-3-phosphate dehydrogenase [GapA], lactate dehydrogenase [Ldh], phosphoglycerate mutase [Pgm], pyruvate kinase [Pyk] and transketolase [Tkt]) to the human extracellular matrix (ECM) proteins fibrinogen (Fn), fibronectin (Fc), lactoferrin (Lf), laminin (Ln) and vitronectin (Vc), respectively. Concentration-dependent interactions between Fn and Vc and all eight recombinant proteins derived from glycolytic enzymes, between Ln and PdhB-C, GapA, Ldh, Pgm, Pyk and Tkt, between Lf and PdhA-C, GapA and Pyk, and between Fc and PdhC and GapA were demonstrated. In most cases, these associations are significantly influenced by ionic forces and by polyclonal sera against recombinant proteins. In immunoblotting, the complex of human plasminogen, activator (tissue-type or urokinase plasminogen activator) and glycolytic enzyme was not able to degrade Fc, Lf and Ln, respectively. In contrast, degradation of Vc was confirmed in the presence of all eight enzymes tested. Our data suggest that the multifaceted associations of surface-localized glycolytic enzymes play a potential role in the adhesion and invasion processes during infection of human respiratory mucosa by M. pneumoniae. Copyright © 2016 Elsevier GmbH. All rights reserved.

Inner clot diffusion and permeation during fibrinolysis.

PubMed Central

Diamond, S L; Anand, S

1993-01-01

A model of fibrinolysis was developed using multicomponent convection-diffusion equations with homogeneous reaction and heterogeneous adsorption and reaction. Fibrin is the dissolving stationary phase and plasminogen, tissue plasminogen activator (tPA), urokinase (uPA), and plasmin are the soluble mobile species. The model is based on an accurate molecular description of the fibrin fiber and protofibril structure and contains no adjustable parameters and one phenomenological parameter estimated from experiment. The model can predict lysis fronts moving across fibrin clots (fine or coarse fibers) of various densities under different administration regimes using uPA and tPA. We predict that pressure-driven permeation is the major mode of transport that allows for kinetically significant thrombolysis during clinical situations. Without permeation, clot lysis would be severely diffusion limited and would require hundreds of minutes. Adsorption of tPA to fibrin under conditions of permeation was a nonequilibrium process that tended to front load clots with tPA. Protein engineering efforts to design optimal thrombolytics will likely be affected by the permeation processes that occur during thrombolysis. PMID:8312497

Quebec platelet disorder.

PubMed

Hayward, Catherine P M; Rivard, Georges E

2011-04-01

Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder associated with a unique gain-of-function defect in fibrinolysis. In the past 5 years, there have been important advances in the understanding of the pathogenesis of QPD, including its genetic cause, which is a copy number variation mutation of PLAU, the gene for urokinase plasminogen activator (uPA). QPD is the first bleeding disorder identified to be caused by a PLAU mutation and it is also the first bleeding disorder recognized to result from a gene copy number mutation. The molecular defect of QPD leads to marked overexpression of uPA during megakaryopoiesis, producing profibrinolytic platelets that contain active forms of uPA in their α-granules. This article summarizes expert opinions on the features of QPD and recent advances in the understanding of its pathogenesis and genetic cause.

[The changes of alpha 2-plasmin inhibitor, and notably urokinase activities in blood measured by synthetic chromogenic substrates S-2444 after urokinase administration].

PubMed

Itoh, Y; Tsuji, K; Tanaka, N; Yamada, M; Nakanishi, M; Ishihara, M; Kobayashi, M

1983-01-01

Thrombolytic therapy with Urokinase (UK) has often been successful but it is very difficult to determine the effective dosage of UK. It is reported that after UK administration, plasmin fibrinolytic activity was immediately inhibited by alpha 2-Plasmin inhibitor (alpha 2-PI). In this study, we used UK on patients with myoma to prevent the occurrence of thrombosis after operation and the initial decrease in alpha 2-PI activities following UK administration was investigated to determine the minimum effective dosage of UK required to suppress alpha 2-PI. An attempt was also made to measure UK activity in blood by means of chromogenic substrate S-2444, and in some cases by administering 60,000 I.U. UK, alpha 1-Antitrypsin (alpha 1-AT), alpha 2-Macroglobulin (alpha 2-M), Antithrombin III (AT III) and Plasminogen (Plg) were measured at the same time. The results were as follows: 1) By the drip infusion method. In all doses, alpha 2-PI and UK activity showed no remarkable change. 2) By the one shot method. a) A decrease in alpha 2-PI was observed following both 48,000 and 60,000 I.U. UK administration. It was noted that in the case of 48,000 I.U. UK, alpha 2-PI showed the lowest level, 60% of the pre-administration level. b) UK activity showed a gradual increase in the case of 60,000 I.U. UK only and large changes in the other cases. c) alpha 1-AT, alpha 2-M, AT III and Plg produced no remarkable changes. This indicated that the effective dosage of UK for suppressing alpha 2-PI was at least 48,000 I.U. UK with the one shot method, and alpha 2-PI is a reliable indicator of the effectiveness of UK therapy.

Vasodilator-Stimulated Phosphoprotein (VASP) depletion from breast cancer MDA-MB-231 cells inhibits tumor spheroid invasion through downregulation of Migfilin, β-catenin and urokinase-plasminogen activator (uPA)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gkretsi, Vasiliki; Stylianou, Andreas; Stylianopoulos, Triantafyllos, E-mail: tstylian@ucy.ac.cy

A hallmark of cancer cells is their ability to invade surrounding tissues and form metastases. Cell-extracellular matrix (ECM)-adhesion proteins are crucial in metastasis, connecting tumor ECM with actin cytoskeleton thus enabling cells to respond to mechanical cues. Vasodilator-stimulated phosphoprotein (VASP) is an actin-polymerization regulator which interacts with cell-ECM adhesion protein Migfilin, and regulates cell migration. We compared VASP expression in MCF-7 and MDA-MB-231 breast cancer (BC) cells and found that more invasive MDA-MB-231 cells overexpress VASP. We then utilized a 3-dimensional (3D) approach to study metastasis in MDA-MB-231 cells using a system that considers mechanical forces exerted by the ECM.more » We prepared 3D collagen I gels of increasing concentration, imaged them by atomic force microscopy, and used them to either embed cells or tumor spheroids, in the presence or absence of VASP. We show, for the first time, that VASP silencing downregulated Migfilin, β-catenin and urokinase plasminogen activator both in 2D and 3D, suggesting a matrix-independent mechanism. Tumor spheroids lacking VASP demonstrated impaired invasion, indicating VASP’s involvement in metastasis, which was corroborated by Kaplan-Meier plotter showing high VASP expression to be associated with poor remission-free survival in lymph node-positive BC patients. Hence, VASP may be a novel BC metastasis biomarker. - Highlights: • More invasive MDA-MB-231 overexpress VASP compared to MCF-7 breast cancer cells. • We prepared 3D collagen I gels of increasing concentration and characterized them. • VASP silencing downregulated Migfilin, β-catenin and uPA both in 2D and 3D culture. • Tumor spheroids lacking VASP demonstrated impaired invasion. • Kaplan-Meier plotter shows association of high VASP expression with poor survival.« less

Urokinase receptor and CXCR4 are regulated by common microRNAs in leukaemia cells

PubMed Central

Alfano, Daniela; Gorrasi, Anna; Li Santi, Anna; Ricci, Patrizia; Montuori, Nunzia; Selleri, Carmine; Ragno, Pia

2015-01-01

The urokinase-type plasminogen activator (uPA) receptor (uPAR) focuses uPA proteolytic activity on the cell membrane, promoting localized degradation of extracellular matrix (ECM), and binds vitronectin (VN), mediating cell adhesion to the ECM. uPAR-bound uPA and VN induce proteolysis-independent intracellular signalling, regulating cell adhesion, migration, survival and proliferation. uPAR cross-talks with CXCR4, the receptor for the stroma-derived factor 1 chemokine. CXCR4 is crucial in the trafficking of hematopoietic stem cells from/to the bone marrow, which involves also uPAR. Both uPAR and CXCR4 are expressed in acute myeloid leukaemia (AML), with a lower expression in undifferentiated and myeloid subsets, and higher expression in myelomonocytic and promyelocytic subsets. We hypothesized a microRNA (miR)-mediated co-regulation of uPAR and CXCR4 expression, which could allow their cross-talk at the cell surface. We identified three miRs, miR-146a, miR-335 and miR-622, regulating the expression of both uPAR and CXCR4 in AML cell lines. Indeed, these miRs directly target the 3′untranslated region of both uPAR- and CXCR4-mRNAs; accordingly, uPAR/CXCR4 expression is reduced by their overexpression in AML cells and increased by their specific inhibitors. Overexpression of all three miRs impairs migration, invasion and proliferation of myelomonocytic cells. Interestingly, we observed an inverse relationship between uPAR/CXCR4 expression and miR-146a and miR-335 levels in AML blasts, suggesting their possible role in the regulation of uPAR/CXCR4 expression also in vivo. PMID:26082201

Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles

PubMed Central

Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

2016-01-01

Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs. PMID:27022410

Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles.

PubMed

Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

2016-01-01

Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs.

Blueberry Phytochemicals Inhibit Growth and Metastatic Potential of MDA-MB-231 Breast Cancer Cells Through Modulation of the Phosphatidylinositol 3-Kinase Pathway

PubMed Central

Adams, Lynn S.; Phung, Sheryl; Yee, Natalie; Seeram, Navindra P.; Li, Liya; Chen, Shiuan

2010-01-01

Dietary phytochemicals are known to exhibit a variety of anti-carcinogenic properties. This study investigated the chemopreventive activity of blueberry extract in triple negative breast cancer cell lines in vitro and in vivo. Blueberry decreased cell proliferation in HCC38, HCC1937 and MDA-MB-231 cells with no effect on the non-tumorigenic MCF-10A cell line. Decreased metastatic potential of MDA-MB-231 cells by blueberry was shown through inhibition of cell motility using wound healing assays and migration through a PET membrane. Blueberry treatment decreased the activity of matrix metalloproteinase 9 and the secretion of urokinase-type plasminogen activator while increasing tissue inhibitor of metalloproteinase-1 and plasminogen activator inhibitor-1 secretion in MDA-MB-231 conditioned medium as shown by western blotting. Cell signaling pathways that control the expression/activation of these processes were investigated via western blotting and reporter gene assay. Treatment with blueberry decreased phosphatidylinositol 3-kinase (PI3K)/AKT and nuclear factor kappa-B (NFκB) activation in MDA-MB-231 cells where protein kinase C (PKC) and extracellular regulated kinase (ERK) were not affected. In vivo, the efficacy of blueberry to inhibit triple negative breast tumor growth was evaluated using the MDA-MB-231 xenograft model. Tumor weight and proliferation (Ki-67 expression) were decreased in blueberry treated mice, where apoptosis (caspase-3 expression) was increased compared to controls. Immunohistochemical analysis of tumors from blueberry-fed mice showed decreased activation of AKT and p65 NFκB signaling proteins with no effect on the phosphorylation of ERK. These data illustrate the inhibitory effect of blueberry phytochemicals on the growth and metastatic potential of MDA-MB-231 cells through modulation of the PI3K/AKT/NFκB pathway. PMID:20388778

Multi-Carotenoids at Physiological Levels Inhibit VEGF-Induced Tube Formation of Endothelial Cells and the Possible Mechanisms of Action Both In Vitro and Ex Vivo.

PubMed

Huang, Chien-Hao; Huang, Chin-Shiu; Hu, Miao-Lin; Chuang, Cheng-Hung

2018-01-01

Carotenoids have been shown to exhibit antiangiogenic activities. Several studies have indicated that carotenoids used in combination were more effective on antioxidation and anticancer actions than carotenoids used singly. However, it is unclear whether multi-carotenoids have antiangiogenic effects. We investigated the effects of multi-carotenoids at physiological plasma levels of Taiwanese (abbreviated as MCT, with a total of 1.4 μM) and Americans (abbreviated as MCA, with a total of 1.8 μM), and of post-supplemental plasma levels (abbreviated as HMC with a total of 3.55 μM) on vascular endothelial growth factor (VEGF)-induced tube formation in human umbilical vein endothelial cells (HUVECs) and rat aortic rings. MCT, MCA, and HMC inhibited VEGF-induced migration, invasion, and tube formation of HUVECs as well as new vessels formation in rat aortic rings. MCT, MCA, and HMC inhibited activities o\\f matrix metalloproteinase (MMP)-2, urokinase plasminogen activator, and phosphorylation of VEGF receptor 2 induced by VEGF. Moreover, MCT, MCA, and HMC significantly upregulated protein expression of tissue inhibitors of MMP-2 and plasminogen activator inhibitor-1. These results demonstrate the antiangiogenic effect of multi-carotenoids both in vitro and ex vivo with possible mechanistic actions involving attenuation of VEGF receptor 2 phosphorylation and extracellular matrix degradation.

Extracellular proteases as targets for drug development

PubMed Central

Cudic, Mare

2015-01-01

Proteases constitute one of the primary targets in drug discovery. In the present review, we focus on extracellular proteases (ECPs) because of their differential expression in many pathophysiological processes, including cancer, cardiovascular conditions, and inflammatory, pulmonary, and periodontal diseases. Many new ECP inhibitors are currently under clinical investigation and a significant increase in new therapies based on protease inhibition can be expected in the coming years. In addition to directly blocking the activity of a targeted protease, one can take advantage of differential expression in disease states to selectively deliver therapeutic or imaging agents. Recent studies in targeted drug development for the metalloproteases (matrix metalloproteinases, adamalysins, pappalysins, neprilysin, angiotensin-converting enzyme, metallocarboxypeptidases, and glutamate carboxypeptidase II), serine proteases (elastase, coagulation factors, tissue/urokinase plasminogen activator system, kallikreins, tryptase, dipeptidyl peptidase IV), cysteine proteases (cathepsin B), and renin system are discussed herein. PMID:19689354

Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto.

PubMed

Yanagisawa, Yasuhide; Chatake, Toshiyuki; Chiba-Kamoshida, Kaori; Naito, Sawa; Ohsugi, Tadanori; Sumi, Hiroyuki; Yasuda, Ichiro; Morimoto, Yukio

2010-12-01

Nattokinase is a single polypeptide chain composed of 275 amino acids (molecular weight 27,724) which displays strong fibrinolytic activity. Moreover, it can activate other fibrinolytic enzymes such as pro-urokinase and tissue plasminogen activator. In the present study, native nattokinase from Bacillus subtilis natto was purified using gel-filtration chromatography and crystallized to give needle-like crystals which could be used for X-ray diffraction experiments. The crystals belonged to space group C2, with unit-cell parameters a=74.3, b=49.9, c=56.3 Å, β=95.2°. Diffraction images were processed to a resolution of 1.74 Å with an Rmerge of 5.2% (15.3% in the highest resolution shell) and a completeness of 69.8% (30.0% in the highest resolution shell). This study reports the first X-ray diffraction analysis of nattokinase.

Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto

PubMed Central

Yanagisawa, Yasuhide; Chatake, Toshiyuki; Chiba-Kamoshida, Kaori; Naito, Sawa; Ohsugi, Tadanori; Sumi, Hiroyuki; Yasuda, Ichiro; Morimoto, Yukio

2010-01-01

Nattokinase is a single polypeptide chain composed of 275 amino acids (molecular weight 27 724) which displays strong fibrinolytic activity. Moreover, it can activate other fibrinolytic enzymes such as pro-urokinase and tissue plasminogen activator. In the present study, native nattokinase from Bacillus subtilis natto was purified using gel-filtration chromatography and crystallized to give needle-like crystals which could be used for X-ray diffraction experiments. The crystals belonged to space group C2, with unit-cell parameters a = 74.3, b = 49.9, c = 56.3 Å, β = 95.2°. Diffraction images were processed to a resolution of 1.74 Å with an R merge of 5.2% (15.3% in the highest resolution shell) and a completeness of 69.8% (30.0% in the highest resolution shell). This study reports the first X-ray diffraction analysis of nattokinase. PMID:21139221

The Effect of Levonorgestrel on Fibrinolytic Factors in Human Endometrial Endothelial Cells.

PubMed

Pakrashi, Tarita; Taylor, Joelle E; Nelson, Ashley; Archer, David F; Jacot, Terry

2016-11-01

The levonorgestrel-releasing intrauterine system is considered a highly effective treatment of heavy menstrual bleeding (HMB). While LNG has established effects on the stromal and glandular compartments of the endometrial tissue, its effect on the endometrial endothelial cells has not been investigated. We examined whether LNG regulates fibrinolytic factors, tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) secreted by human endometrial endothelial cells (HEECs) and determined the steroid receptor through which LNG exerts its effect on the endothelium. The HEECs were treated with LNG or progesterone and levels of tPA and plasminogen activator inhibitor 1 (PAI-1) measured. The HEECs were specifically examined for the presence of androgen receptors through Western blot. Levonorgestrel ± flutamide were added to HEECs and the levels of tPA and uPA were examined. An enzyme-linked immunosorbent assay performed on culture media confirmed a statistically significant decrease in tPA levels in cells treated with LNG (77.80% ± 8.0% of control; n = 5, P < .05 vs control) but not progesterone. The androgen receptor (110 kDa) was detected in HEEC lysates. The decrease in tPA was blocked by the addition of flutamide (101.3% ± 16% of control), a classic nonsteroidal androgen receptor blocker. There was no change in uPA or PAI-1 levels in cells treated with LNG. Levonorgestrel decreases tPA levels through the androgen receptor in HEECs. Thus, LNG inhibits tPA secretion by the endometrial endothelial cell. This response suggests reduction in HMB with LNG-IUS could reflect an LNG-mediated promotion of hemostasis. © The Author(s) 2016.

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

PubMed

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-02-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

PubMed Central

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-01-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators. Images PMID:3257578

Enhancement of cutaneous wound healing by Dsg2 augmentation of uPAR secretion.

PubMed

Cooper, Felicia; Overmiller, Andrew M; Loder, Anthony; Brennan-Crispi, Donna M; McGuinn, Kathleen P; Marous, Molly R; Freeman, Theresa A; Riobo-Del Galdo, Natalia A; Siracusa, Linda D; Wahl, James K; Mahoney, Mỹ G

2018-05-09

In addition to playing a role in adhesion, desmoglein 2 (Dsg2) is an important regulator of growth and survival signaling pathways, cell proliferation, migration and invasion, and oncogenesis. While low-level Dsg2 expression is observed in basal keratinocytes and is downregulated in non-healing venous ulcers, overexpression has been observed in both melanomas and non-melanoma malignancies. Here, we show that transgenic mice overexpressing Dsg2 in basal keratinocytes primed the activation of mitogenic pathways, but did not induce dramatic epidermal changes or susceptibility to chemical-induced tumor development. Interestingly, acceleration of full-thickness wound closure and increased wound-adjacent keratinocyte proliferation was observed in these mice. As epidermal cytokines and their receptors play critical roles in wound healing, Dsg2-induced secretome alterations were assessed with an antibody profiler array and revealed increased release and proteolytic processing of the urokinase-type plasminogen activator receptor (uPAR). Dsg2 induced uPAR expression in the skin of transgenic compared to wild-type mice. Wound healing further enhanced uPAR in both epidermis and dermis with concomitant increase in the pro-healing laminin-332, a major component of the basement membrane zone, in transgenic mice. This study demonstrates that Dsg2 induces epidermal activation of various signaling cascades and accelerates cutaneous wound healing, in part, through uPAR-related signaling cascades. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Tristetraprolin: A novel target of diallyl disulfide that inhibits the progression of breast cancer.

PubMed

Xiong, Ting; Liu, Xiao-Wang; Huang, Xue-Long; Xu, Xiong-Feng; Xie, Wei-Quan; Zhang, Su-Jun; Tu, Jian

2018-05-01

Diallyl disulfide (DADS), a volatile component of garlic oil, has various biological properties, including antioxidant, antiangiogenic and anticancer effects. The present study aimed to explore novel targets of DADS that may slow or stop the progression of breast cancer. First, xenograft tumor models were created by subcutaneously injecting MCF-7 and MDA-MB-231 breast cancer cells into nude mice. Subsequently, western blot analysis was performed to investigate the expression of tristetraprolin (TTP), urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) in the xenograft tumors, and cell cultures. Tablet cloning, Transwell and wound healing assays revealed that DADS treatment significantly inhibited the proliferation, invasion and migration of breast cancer cells. In addition, DADS treatment led to significant downregulation of uPA and MMP-9 protein expression, but significantly upregulated TTP expression in vivo and in vitro . Knocking down TTP expression using small interfering RNA reversed the aforementioned effects of DADS, which suggests TTP is a key target of DADS in inhibiting the progression of breast cancer.

Nanomechanical recognition of prognostic biomarker suPAR with DVD-ROM optical technology.

PubMed

Bache, Michael; Bosco, Filippo G; Brøgger, Anna L; Frøhling, Kasper B; Alstrøm, Tommy Sonne; Hwu, En-Te; Chen, Ching-Hsiu; Eugen-Olsen, Jesper; Hwang, Ing-Shouh; Boisen, Anja

2013-11-08

In this work the use of a high-throughput nanomechanical detection system based on a DVD-ROM optical drive and cantilever sensors is presented for the detection of urokinase plasminogen activator receptor inflammatory biomarker (uPAR). Several large scale studies have linked elevated levels of soluble uPAR (suPAR) to infectious diseases, such as HIV, and certain types of cancer. Using hundreds of cantilevers and a DVD-based platform, cantilever deflection response from antibody-antigen recognition is investigated as a function of suPAR concentration. The goal is to provide a cheap and portable detection platform which can carry valuable prognostic information. In order to optimize the cantilever response the antibody immobilization and unspecific binding are initially characterized using quartz crystal microbalance technology. Also, the choice of antibody is explored in order to generate the largest surface stress on the cantilevers, thus increasing the signal. Using optimized experimental conditions the lowest detectable suPAR concentration is currently around 5 nM. The results reveal promising research strategies for the implementation of specific biochemical assays in a portable and high-throughput microsensor-based detection platform.

M2-like macrophages are responsible for collagen degradation through a mannose receptor–mediated pathway

PubMed Central

Madsen, Daniel H.; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E.; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S.; Brenner, David A.; Burgdorf, Sven; Engelholm, Lars H.; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto

2013-01-01

Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase–dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor–associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process. PMID:24019537

Osteopontin ablation ameliorates muscular dystrophy by shifting macrophages to a pro-regenerative phenotype

PubMed Central

Capote, Joana; Martinez, Leonel; Vetrone, Sylvia; Barton, Elisabeth R.; Sweeney, H. Lee; Miceli, M. Carrie

2016-01-01

In the degenerative disease Duchenne muscular dystrophy, inflammatory cells enter muscles in response to repetitive muscle damage. Immune factors are required for muscle regeneration, but chronic inflammation creates a profibrotic milieu that exacerbates disease progression. Osteopontin (OPN) is an immunomodulator highly expressed in dystrophic muscles. Ablation of OPN correlates with reduced fibrosis and improved muscle strength as well as reduced natural killer T (NKT) cell counts. Here, we demonstrate that the improved dystrophic phenotype observed with OPN ablation does not result from reductions in NKT cells. OPN ablation skews macrophage polarization toward a pro-regenerative phenotype by reducing M1 and M2a and increasing M2c subsets. These changes are associated with increased expression of pro-regenerative factors insulin-like growth factor 1, leukemia inhibitory factor, and urokinase-type plasminogen activator. Furthermore, altered macrophage polarization correlated with increases in muscle weight and muscle fiber diameter, resulting in long-term improvements in muscle strength and function in mdx mice. These findings suggest that OPN ablation promotes muscle repair via macrophage secretion of pro-myogenic growth factors. PMID:27091452

C-Terminal Truncation of α-COP Affects Functioning of Secretory Organelles and Calcium Homeostasis in Hansenula polymorpha

PubMed Central

Chechenova, Maria B.; Romanova, Nina V.; Deev, Alexander V.; Packeiser, Anna N.; Smirnov, Vladimir N.; Agaphonov, Michael O.; Ter-Avanesyan, Michael D.

2004-01-01

In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport. Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding α-COP, a subunit of the COPI protein complex. H. polymorpha ret1 mutants, which expressed truncated α-COP lacking more than 300 C-terminal amino acids, manifested an enhanced ability to secrete human urokinase-type plasminogen activator (uPA) and an inability to grow with a shortage of Ca2+ ions, whereas a lack of α-COP expression was lethal. The α-COP defect also caused alteration of intracellular transport of the glycosylphosphatidylinositol-anchored protein Gas1p, secretion of abnormal uPA forms, and reductions in the levels of Pmr1p, a Golgi Ca2+-ATPase. Overexpression of Pmr1p suppressed some ret1 mutant phenotypes, namely, Ca2+ dependence and enhanced uPA secretion. The role of COPI-dependent vesicular transport in cellular Ca2+ homeostasis is discussed. PMID:14871936

Characterization of two new putative adhesins of Leptospira interrogans.

PubMed

Figueredo, Jupciana M; Siqueira, Gabriela H; de Souza, Gisele O; Heinemann, Marcos B; Vasconcellos, Silvio A; Chapola, Erica G B; Nascimento, Ana L T O

2017-01-01

We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and a hypothetical protein. The CDSs LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasmic membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix components revealed that they are laminin binding and they were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen and generate plasmin in the presence of urokinase-type plasminogen activator. Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the cell surface playing a secondary role, probably mediating host -pathogen interaction, suggests that LIC11122 is a moonlighting protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest that Lsa19 is a potential candidate for future evaluation of its role in adhesion/colonization activities during L. interrogans infection.

Modulation of serpin reaction through stabilization of transient intermediate by ligands bound to alpha-helix F.

PubMed

Komissarov, Andrey A; Zhou, Aiwu; Declerck, Paul J

2007-09-07

Mechanism-based inhibition of proteinases by serpins involves enzyme acylation and fast insertion of the reactive center loop (RCL) into the central beta-sheet of the serpin, resulting in mechanical inactivation of the proteinase. We examined the effects of ligands specific to alpha-helix F (alphaHF) of plasminogen activator inhibitor-1 (PAI-1) on the stoichiometry of inhibition (SI) and limiting rate constant (k(lim)) of RCL insertion for reactions with beta-trypsin, tissue-type plasminogen activator (tPA), and urokinase. The somatomedin B domain of vitronectin (SMBD) did not affect SI for any proteinase or k(lim) for tPA but decreased the k(lim) for beta-trypsin. In contrast to SMBD, monoclonal antibodies MA-55F4C12 and MA-33H1F7, the epitopes of which are located at the opposite side of alphaHF, decreased k(lim) and increased SI for every enzyme. These effects were enhanced in the presence of SMBD. RCL insertion for beta-trypsin and tPA is limited by different subsequent steps of PAI-1 mechanism as follows: enzyme acylation and formation of a loop-displaced acyl complex (LDA), respectively. Stabilization of LDA through the disruption of the exosite interactions between PAI-1 and tPA induced an increase in the k(lim) but did not affect the SI. Thus it is unlikely that LDA contributes significantly to the outcome of the serpin reaction. These results demonstrate that the rate of RCL insertion is not necessarily correlated with SI and indicate that an intermediate, different from LDA, which forms during the late steps of PAI-1 mechanism, and could be stabilized by ligands specific to alphaHF, controls bifurcation between the inhibitory and the substrate pathways.

Be Active or Not: the Relative Contribution of Active and Passive Tumor Targeting of Nanomaterials

PubMed Central

Li, Rui; Zheng, Ke; Yuan, Cai; Chen, Zhuo; Huang, Mingdong

2017-01-01

Malignant tumor (cancer) remains as one of the deadliest diseases throughout the world, despite its overall mortality drops. Nanomaterials (NMs) have been widely studied as diagnostic and/or therapeutic agents for tumors. A feature of NMs, compared to small molecules, is that NMs can be concentrated passively in tumors through enhanced permeability and retention (EPR) effect. In the meantime, NMs can be engineered to target toward tumor specific markers in an active manner, e.g., receptor-mediated targeting. The relative contribution of the EPR effect and the receptor-mediated targeting to NM accumulation in tumor tissues has not been clearly defined yet. Here, we tackle this fundamental issue by reviewing previous studies. First, we summarize the current knowledge on these two tumor targeting strategies of NMs, and on how NMs arrive to tumors from blood circulation. We then demonstrate that contribution of the active and passive effects to total accumulation of NMs in tumors varies with time. Over time, the receptor-mediated targeting contributes more than the EPR effect with a ratio of 3 in the case of urokinase-type plasminogen activator receptor (uPAR)-mediated targeting and human serum albumin (HSA)-mediated EPR effect. Therefore, this review highlights the dynamics of active and passive targeting of NMs on their accumulation at tumor sites, and is valuable for future design of NMs in cancer diagnosis and treatment. PMID:29071198

Mechanisms regulating plasminogen activators in transformed retinal ganglion cells

PubMed Central

Rock, Nathan; Chintala, Shravan K.

2008-01-01

Irreversible loss of retinal ganglion cells (RGCs) is a major clinical issue in glaucoma, but the mechanisms that lead to RGC death are currently unclear. We have previously reported that elevated levels of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) cause the death of RGCs in vivo and transformed retinal ganglion cells (RGC-5) in vitro. Yet, it is unclear how secreted proteases such as tPA and uPA directly cause RGCs' death. In this study, by employing RGC-5 cells, we report that tPA and uPA elicit their direct effect through the low-density lipoprotein-related receptor-1 (LRP-1). We also show that blockade of protease-LRP-1 interaction leads to a compete reduction in autocrine synthesis of tPA and uPA, and prevents protease-mediated death of RGC-5 cells. RGC-5 cells were cultured in serum-free medium and treated with 2.0 uM Staurosporine to induce their differentiation. Neurite outgrowth was observed by a phase contrast microscope and quantified by NeuroJ imaging software. Proteolytic activities of tPA and uPA were determined by zymography assays. Cell viability was determined by MTT assays. Compared to untreated RGC-5 cells, cells treated with Staurosporine differentiated, synthesized and secreted elevated levels of tPA and uPA, and underwent cell death. In contrast, when RGC-5 cells were treated with Staurosporine along with the receptor associated protein (RAP), proteolytic activities of both tPA and uPA were significantly reduced. Under these conditions, a significant number of RGC-5 cells survived and showed increased neurite outgrowth. These results indicate that LRP-1 regulates autocrine synthesis of tPA and uPA in RGC-5 cells and suggest that the use of RAP to antagonize the effect of proteases may be a way to prevent RGC death in glaucoma. PMID:18243176

Krüppel-like factor 17 inhibits urokinase plasminogen activator gene expression to suppress cell invasion through the Src/p38/MAPK signaling pathway in human lung adenocarcinoma

PubMed Central

Huang, Shuai; Li, Jiong; Liu, Xiao-Yan; Pan, Xing-Fei; Wang, Qin-Qin; Chen, Li; Lin, Ming-Juan; Huang, Zhi-Hong; Ma, Hong-Ming; Wu, Yi; Liu, Sheng-Ming; Zhou, Yan-Bin

2017-01-01

Krüppel-like factor 17 (KLF17) has been reported to be involved in invasion and metastasis suppression in lung cancer, but the molecular mechanisms underlying the anti-invasion and anti-metastasis roles of KLF17 in lung cancer are not fully illustrated. Here, we showed that KLF17 inhibited the invasion of A549 and H322 cells; the anti-invasion effect of KLF17 was associated with the suppression of urokinase plasminogen activator (uPA/PLAU) expression. KLF17 can bind with the promoter of uPA and inhibit its expression. Enforced expression of uPA abrogated the anti-invasion effect of KLF17 in A549 and H322 cells. In addition, immunohistochemistry staining showed that the protein expression of KLF17 was negatively correlated with that of uPA in archived samples from patients with lymph node metastasis of lung adenocarcinoma (rho = −0.62, P = 0.01). The mutually exclusive expression of KLF17 with uPA could predict lymph node metastasis for lung adenocarcinoma (AUC = 0.758, P = 0.005). Enforced expression of KLF17 inhibited the expression of phosphorylated Src and phosphorylated p38/MAPK in A549 and H322 cells. The invasiveness of the cells were suppressed by treating with sb203580 (p38/MAPK inhibitor) or HY-13805 (PP2, Src inhibitor). furthermore, p38/MAPK inhibition could block the KLF17-induced reduction of p-p38/MAPK and uPA, and Src inhibition enhanced the KLF17-induced suppression of p-Src and uPA in A549 and H322 cells. In conclusion, our study indicated that KLF17 suppressed the uPA-mediated invasion of lung adenocarcinoma. The Src and p38/MAPK signaling pathways were suggested as mediators of KLF17-induced uPA inhibition, thus providing evidence that KLF17 might be a potential anti-invasion candidate for lung adenocarcinoma. PMID:28454121

Urokinase and its Receptors in Chronic Kidney Disease

PubMed Central

Zhang, Guoqiang; Eddy, Allison A.

2011-01-01

Since the recognition that plasminogen activator inhibitor-1 (PAI-1) is a powerful profibrotic molecule, there has been considerable interest in deciphering the extent to which this effect is mediated by its ability to inhibit serine proteases with downstream effects on fibrogenesis. This review will summarize current knowledge about the serine protease urokinase-type plasminogen activator and its high affinity receptor uPAR/CD87 as it pertains to chronic kidney disease (CKD) progression. An emerging theme is that the effects of PAI-1 and uPAR appear to be organ- and site-specific. Normal kidney tubules produce a large quantity of uPA that is secreted into the urinary space. Activity levels increase during CKD presumably due to new sources of production by macrophages and fibroblasts. By activating hepatocyte growth factor and degrading fibrinogen uPA may have anti-fibrotic effects. However CKD severity after experimental ureteral obstruction is not altered by endogenous uPA deficiency. Beneficial effects of exogenous uPA have been reported in experimental models of fibrosis in the lung and liver but CKD awaits exploration. Absent in normal kidneys uPAR is expressed by both renal parenchymal cells and inflammatory cells in a variety of pathological states. Such expression appears beneficial based on studies performed in uPAR-deficient mice. The uPAR promotes bacterial clearance in infectious diseases. In CKD uPAR expression is associated with high uPA activity but its most important effect appears to be due to scavenging activities and effects on cell recruitment and migration. Although uPAR itself is a non-signaling receptor, it interacts with a variety of co-receptors to modify cellular behavior. Best known are interactions with the low-density lipoprotein receptor-related protein (LRP-1) that lead to PAI-1 endocytosis and degradation, and interactions with several integrins to regulate matrix-dependent cell migration. Contacts with the receptor for the complement C5a component and the interleukin −6 receptor gp130 are examples of other recently recognized interactions. In addition to uPA, vitronectin and high molecular weight kininogen are alternate uPAR ligands that could be implicated in CKD progression. uPAR may also be shed from cell membranes. This soluble form (suPAR) has been detected in plasma and urine and is known to be a chemoattractant for leukocytes that express the formyl-peptide-receptor-like receptor 1/lipoxin A4 receptor. In addition to uPAR several other receptors, including some of the uPAR co-receptors, may also bind directly to uPA and activate cell signaling pathways. The roles of these newer uPAR ligands and uPA receptors are just beginning to be investigated. Since many of them are expressed in the kidney, their potential participation in CKD pathogenesis will be of interest. PMID:18508599

Mesenchymal stem cells from adipose and bone marrow promote angiogenesis via distinct cytokine and protease expression mechanisms

PubMed Central

Kachgal, Suraj; Putnam, Andrew J.

2012-01-01

Using a fibrin-based angiogenesis model, we have established that there is no canonical mechanism used by ECs to degrade the surrounding extracellular matrix (ECM), but rather the set of proteases used is dependent on the mural cells providing the angiogenic cues. Mesenchymal stem cells (MSCs) originating from different tissues, which are thought to be phenotypically similar, promote angiogenesis through distinct mechanisms. Specifically, adipose-derived stem cells (ASCs) promote utilization of the plasminogen activator-plasmin axis by ECs as the primary means of vessel invasion and elongation in fibrin. Matrix metalloproteinases (MMPs) serve a purpose in regulating capillary diameter and possibly in stabilizing the nascent vessels. These proteolytic mechanisms are more akin to those involved in fibroblast-mediated angiogenesis than to those in bone marrow-derived stem cell (BMSC)-mediated angiogenesis. In addition, expression patterns of angiogenic factors such as urokinase plasminogen activator (uPA), hepatocyte growth factor (HGF), and tumor necrosis factor alpha (TNFα) were similar for ASC and fibroblast-mediated angiogenesis, and in direct contrast to BMSC-mediated angiogenesis. The present study illustrates that the nature of the heterotypic interactions between mural cells and endothelial cells depend on the identity of the mural cell used. Even MSCs which are shown to behave phenotypically similar do not stimulate angiogenesis via the same mechanisms. PMID:21104120

MicroRNA-23b mediates urokinase and c-met downmodulation and a decreased migration of human hepatocellular carcinoma cells.

PubMed

Salvi, Alessandro; Sabelli, Cristiano; Moncini, Silvia; Venturin, Marco; Arici, Bruna; Riva, Paola; Portolani, Nazario; Giulini, Stefano M; De Petro, Giuseppina; Barlati, Sergio

2009-06-01

Urokinase-type plasminogen activator (uPA) and c-met play a major role in cancer invasion and metastasis. Evidence has suggested that uPA and c-met overexpression may be coordinated in human hepatocellular carcinoma (HCC). In the present study, to understand whether the expression of these genes might be coregulated by specific microRNAs (miRs) in human cells, we predicted that Homo sapiens microRNA-23b could recognize two sites in the 3'-UTR of uPA and four sites in the c-met 3'-UTR by the algorithm pictar. The miR-23b expression analysis in human tumor and normal cells revealed an inverse trend with uPA and c-met expression, indicating that uPA and c-met negative regulation might depend on miR-23b expression. Transfection of miR-23b molecules in HCC cells (SKHep1C3) led to inhibition of protein expression of the target genes and caused a decrease in cell migration and proliferation capabilities. Furthermore, anti-miR-23b transfection in human normal AB2 dermal fibroblasts upregulated the expression of endogenous uPA and c-met. Cotransfection experiments in HCC cells of the miR-23b with pGL4.71 Renilla luciferase reporter gene constructs, containing the putative uPA and c-met 3'-UTR target sites, and with the pGL3 firefly luciferase-expressing vector showed a decrease in the relative luciferase activity. This would indicate that miR-23b can recognize target sites in the 3'-UTR of uPA and of c-met mRNAs and translationally repress the expression of uPA and c-met in HCC cells. The evidence obtained shows that overexpression of miR-23b leads to uPA and c-met downregulation and to decreased migration and proliferation abilities of HCC cells.

Metabolic factors, adipose tissue, and plasminogen activator inhibitor-1 levels in Type 2 diabetes

USDA-ARS?s Scientific Manuscript database

Plasminogen activator inhibitor-1 (PAI-1) production by adipose tissue is increased in obesity, and its circulating levels are high in type 2 diabetes. PAI-1 increases cardiovascular risk by favoring clot stability, interfering with vascular remodeling, or both. We investigated in obese diabetic per...

Urokinase and the intestinal mucosa: evidence for a role in epithelial cell turnover

PubMed Central

Gibson, P; Birchall, I; Rosella, O; Albert, V; Finch, C; Barkla, D; Young, G

1998-01-01

Background—The functions of urokinase in intestinal epithelia are unknown. 
Aims—To determine the relation of urokinase expressed by intestinal epithelial cells to their position in the crypt-villus/surface axis and of mucosal urokinase activity to epithelial proliferative kinetics in the distal colon. 
Methods—Urokinase expression was examined immunohistochemically in human intestinal mucosa. Urokinase activity was measured colorimetrically in epithelial cells isolated sequentially from the crypt-villus axis of the rat small intestine. In separate experiments, urokinase activity and epithelial kinetics (measured stathmokinetically) were measured in homogenates of distal colonic mucosa of 14 groups of eight rats fed diets known to alter epithelial turnover. 
Results—From the crypt base, an ascending gradient of expression and activity of urokinase was associated with the epithelial cells. Median mucosal urokinase activities in each of the dietary groups of rats correlated positively with autologous median number of metaphase arrests per crypt (r=0.68; p<0.005) and per 100 crypt cells (r=0.75; p<0.001), but not with crypt column height. 
Conclusions—Localisation of an enzyme capable of leading to digestion of cell substratum in the region where cells are loosely attached to their basement membrane, and the association of its activity with indexes of cell turnover, suggest a role for urokinase in facilitating epithelial cell loss in the intestine. 

 Keywords: urokinase; intestinal epithelium; colon; epithelial proliferation PMID:9824347

Anti-proliferative Effect of Engineered Neural Stem Cells Expressing Cytosine Deaminase and Interferon-β against Lymph Node–Derived Metastatic Colorectal Adenocarcinoma in Cellular and Xenograft Mouse Models

PubMed Central

Park, Geon-Tae; Kim, Seung U.; Choi, Kyung-Chul

2017-01-01

Purpose Genetically engineered stem cells may be advantageous for gene therapy against various human cancers due to their inherent tumor-tropic properties. In this study, genetically engineered human neural stem cells (HB1.F3) expressing Escherichia coli cytosine deaminase (CD) (HB1.F3.CD) and human interferon-β (IFN-β) (HB1.F3.CD.IFN-β) were employed against lymph node–derived metastatic colorectal adenocarcinoma. Materials and Methods CD can convert a prodrug, 5-fluorocytosine (5-FC), to active 5-fluorouracil, which inhibits tumor growth through the inhibition of DNA synthesis,while IFN-β also strongly inhibits tumor growth by inducing the apoptotic process. In reverse transcription polymerase chain reaction analysis, we confirmed that HB1.F3.CD cells expressed the CD gene and HB1.F3.CD.IFN-β cells expressed both CD and IFN-β genes. Results In results of a modified trans-well migration assay, HB1.F3.CD and HB1.F3.CD.IFN-β cells selectively migrated toward SW-620, human lymph node–derived metastatic colorectal adenocarcinoma cells. The viability of SW-620 cells was significantly reduced when co-cultured with HB1.F3.CD or HB1.F3.CD.IFN-β cells in the presence of 5-FC. In addition, it was found that the tumor-tropic properties of these engineered human neural stem cells (hNSCs) were attributed to chemoattractant molecules including stromal cell-derived factor 1, c-Kit, urokinase receptor, urokinase-type plasminogen activator, and C-C chemokine receptor type 2 secreted by SW-620 cells. In a xenograft mouse model, treatment with hNSC resulted in significantly inhibited growth of the tumor mass without virulent effects on the animals. Conclusion The current results indicate that engineered hNSCs and a prodrug treatment inhibited the growth of SW-620 cells. Therefore, hNSC therapy may be a clinically effective tool for the treatment of lymph node metastatic colorectal cancer. PMID:27188205

Plasminogen stimulates propagation of protease-resistant prion protein in vitro.

PubMed

Mays, Charles E; Ryou, Chongsuk

2010-12-01

To clarify the role of plasminogen as a cofactor for prion propagation, we conducted functional assays using a cell-free prion protein (PrP) conversion assay termed protein misfolding cyclic amplification (PMCA) and prion-infected cell lines. Here, we report that plasminogen stimulates propagation of the protease-resistant scrapie PrP (PrP(Sc)). Compared to control PMCA conducted without plasminogen, addition of plasminogen in PMCA using wild-type brain material significantly increased PrP conversion, with an EC(50) = ∼56 nM. PrP conversion in PMCA was substantially less efficient with plasminogen-deficient brain material than with wild-type material. The activity stimulating PrP conversion was specific for plasminogen and conserved in its kringle domains. Such activity was abrogated by modification of plasminogen structure and interference of PrP-plasminogen interaction. Kinetic analysis of PrP(Sc) generation demonstrated that the presence of plasminogen in PMCA enhanced the PrP(Sc) production rate to ∼0.97 U/μl/h and reduced turnover time to ∼1 h compared to those (∼0.4 U/μl/h and ∼2.5 h) obtained without supplementation. Furthermore, as observed in PMCA, plasminogen and kringles promoted PrP(Sc) propagation in ScN2a and Elk 21(+) cells. Our results demonstrate that plasminogen functions in stimulating conversion processes and represents the first cellular protein cofactor that enhances the hypothetical mechanism of prion propagation.

Structural Principles in the Development of Cyclic Peptidic Enzyme Inhibitors

PubMed Central

Xu, Peng; Andreasen, Peter A.; Huang, Mingdong

2017-01-01

This review summarizes our studies in the development of small cyclic peptides for specifically modulating enzyme activity. Serine proteases share highly similar active sites but perform diverse physiological and pathological functions. From a phage-display peptide library, we isolated two mono-cyclic peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), which inhibit the activity of human and murine urokinase-type plasminogen activators (huPA and muPA) with Ki values in the micromolar or sub-micromolar range, respectively. The following affinity maturations significantly enhanced the potencies of the two peptides, 10-fold and >250-fold for upain-1 and mupain-1, respectively. The most potent muPA inhibitor has a potency (Ki = 2 nM) and specificity comparable to mono-clonal antibodies. Furthermore, we also found an unusual feature of mupain-1 that its inhibitory potency can be enhanced by increasing the flexibility, which challenges the traditional viewpoint that higher rigidity leading to higher affinity. Moreover, by changing a few key residues, we converted mupain-1 from a uPA inhibitor to inhibitors of other serine proteases, including plasma kallikrein (PK) and coagulation factor XIa (fXIa). PK and fXIa inhibitors showed Ki values in the low nanomolar range and high specificity. Our studies demonstrate the versatility of small cyclic peptides to engineer inhibitory potency against serine proteases and to provide a new strategy for generating peptide inhibitors of serine proteases. PMID:29104489

A superactive leptin antagonist alters metabolism and locomotion in high-leptin mice.

PubMed

Chapnik, Nava; Solomon, Gili; Genzer, Yoni; Miskin, Ruth; Gertler, Arieh; Froy, Oren

2013-06-01

Transgenic alpha murine urokinase-type plasminogen activator (αMUPA) mice are resistant to obesity and their locomotor activity is altered. As these mice have high leptin levels, our objective was to test whether leptin is responsible for these characteristics. αMUPA, their genetic background control (FVB/N), and C57BL mice were injected s.c. every other day with 20  mg/kg pegylated superactive mouse leptin antagonist (PEG-SMLA) for 6 weeks. We tested the effect of PEG-SMLA on body weight, locomotion, and bone health. The antagonist led to a rapid increase in body weight and subsequent insulin resistance in all treated mice. Food intake of PEG-SMLA-injected animals increased during the initial period of the experiment but then declined to a similar level to that of the control animals. Interestingly, αMUPA mice were found to have reduced bone volume (BV) than FVB/N mice, although PEG-SMLA increased bone mass in both strains. In addition, PEG-SMLA led to disrupted locomotor activity and increased corticosterone levels in C57BL but decreased levels in αMUPA or FVB/N mice. These results suggest that leptin is responsible for the lean phenotype and reduced BV in αMUPA mice; leptin affects corticosterone levels in mice in a strain-specific manner; and leptin alters locomotor activity, a behavior determined by the central circadian clock.

Loss or Inhibition of uPA or MMP-9 Attenuates LV Remodeling and Dysfunction after Acute Pressure Overload in Mice

PubMed Central

Heymans, Stephane; Lupu, Florea; Terclavers, Sven; Vanwetswinkel, Bjorn; Herbert, Jean-Marc; Baker, Andrew; Collen, Desire; Carmeliet, Peter; Moons, Lieve

2005-01-01

Left ventricular (LV) hypertrophy is a natural response of the heart to increased pressure loading, but accompanying fibrosis and dilatation may result in irreversible life-threatening heart failure. Matrix metalloproteinases (MMPs) have been invoked in various cardiac diseases, however, direct genetic evidence for a role of the plasminogen activator (PA) and MMP systems in pressure overload-induced LV hypertrophy and in heart failure is lacking. Therefore, the consequences of transverse aortic banding (TAB) were analyzed in mice lacking tissue-type PA (t-PA−/−), urokinase-type PA (u-PA−/−), or gelatinase-B (MMP-9−/−), and in wild-type (WT) mice after adenoviral gene transfer of the PA-inhibitor PAI-1 or the MMP-inhibitor TIMP-1. TAB elevated LV pressure comparably in all genotypes. In WT and t-PA−/− mice, cardiomyocyte hypertrophy was associated with myocardial fibrosis, LV dilatation and dysfunction, and pump failure after 7 weeks. In contrast, in u-PA−/− mice or in WT mice after PAI-1- and TIMP-1-gene transfer, cardiomyocyte hypertrophy was moderate and only minimally associated with cardiac fibrosis and LV dilatation, resulting in better preservation of pump function. Deficiency of MMP-9 had an intermediate effect. These findings suggest that the use of u-PA- or MMP-inhibitors might preserve cardiac pump function in LV pressure overloading. PMID:15631996

Supermacroporous cryogel matrix for integrated protein isolation. Immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line.

PubMed

Kumar, Ashok; Bansal, Vibha; Andersson, Jonatan; Roychoudhury, Pradip K; Mattiasson, Bo

2006-01-20

A new type of supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing the specific capture of urokinase from conditioned media of human fibrosarcoma cell line HT1080. The affinity adsorbent was designed with the objective of using it as a capture column in an integrated perfusion/protein separation bioreactor setup. A comparative study between the utility of this novel cryogel based matrix and the conventional Sepharose based affinity matrix for the continuous capture of urokinase in an integrated bioreactor system was performed. Cu(II)-ion was coupled to epoxy activated polyacrylamide cryogel and Sepharose using iminodiacetic acid (IDA) as the chelating ligand. About 27-fold purification of urokinase from the conditioned culture media was achieved with Cu(II)-IDA-polyacrylamide cryogel column giving specific activity of about 814 Plough units (PU)/mg protein and enzyme yields of about 80%. High yields (95%) were obtained with Cu(II)-IDA-Sepharose column by virtue of its high binding capacity. However, the adsorbent showed lower selectivity as compared to cryogel matrix giving specific activity of 161 PU/mg protein and purification factor of 5.3. The high porosity, selectivity and reasonably good binding capacity of Cu(II)-IDA-polyacrylamide cryogel column make it a promising option for use as a protein capture column in integrated perfusion/separation processes. The urokinase peak pool from Cu(II)-IDA-polyacrylamide cryogel column could be further resolved into separate fractions for high and low molecular weight forms of urokinase by gel filtration chromatography on Sephacryl S-200. The selectivity of the cryogel based IMAC matrix for urokinase was found to be higher as compared to that of Cu(II)-IDA-Sepharose column.

Inhibitory effect of vitamin C in combination with vitamin K3 on tumor growth and metastasis of Lewis lung carcinoma xenografted in C57BL/6 mice.

PubMed

Chen, Ming-Feng; Yang, Chih-Min; Su, Cheng-Ming; Liao, Jiunn-Wang; Hu, Miao-Lin

2011-01-01

Vitamin C in combination with vitamin K3 (vit CK3) has been shown to inhibit tumor growth and lung metastasis in vivo, but the mechanism of action is poorly understood. Herein, C57BL/6 mice were implanted (s.c.) with Lewis lung carcinoma (LLC) for 9 days before injection (i.p.) with low-dose (100 mg vit C/kg + 1 mg vit K3/kg), high-dose (1,000 mg vit C/kg + 10 mg vit K3/kg) vit CK3 twice a week for an additional 28 days. As expected, vit CK3 or cisplatin (6 mg/kg, as a positive control) significantly and dose-dependently inhibited tumor growth and lung metastasis in LLC-bearing mice. Vit CK3 restored the body weight of tumor-bearing mice to the level of tumor-free mice. Vit CK3 significantly decreased activities of plasma metalloproteinase (MMP)-2, -9, and urokinase plasminogen activator (uPA). In lung tissues, vit CK3 1) increased protein expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2, nonmetastatic protein 23 homolog 1 and plasminogen activator inhibitor-1; 2) reduced protein expression of MMP-2 and MMP-9; and 3) inhibited the proliferating cell nuclear antigen (PCNA). These results demonstrate that vit CK3 inhibits primary tumor growth and exhibits antimetastastic potential in vivo through attenuated tumor invasion and proliferation.

Genistein modifies liver fibrosis and improves liver function by inducing uPA expression and proteolytic activity in CCl4-treated rats.

PubMed

Salas, Alfonso Leija; Montezuma, Tania Díaz; Fariña, German Garrido; Reyes-Esparza, Jorge; Rodríguez-Fragoso, Lourdes

2008-01-01

To evaluate the effect of genistein on the fibrosis and matrix degradation caused by experimentally induced fibrosis in rats. Hepatic fibrosis was brought about by chronic administration of carbon tetrachloride to rats. To evaluate the effect of genistein on liver fibrosis and function, total collagen content and proteolytic activity in the liver were quantified. Urokinase-type plasminogen activator (uPA) expression during experimental fibrosis was localized by immunohistochemistry. Histopathological changes were evaluated using light and electron microscopy. Animals with fibrosis and treated with genistein showed an important reduction (73%) in hepatic collagen content as well as an improvement in liver function (p < 0.001). Genistein increased the capacity of the liver to degrade type I collagen and Matrigel (3.1- and 3.7-fold, respectively; p < 0.001) in animals with liver fibrosis. Genistein increased the number of uPA-immunoreactive cells. The increase in the uPA expression correlated with an increase in proteolytic activity. Histological analysis revealed a reduction in the number of fiber septa in pericentral and perisinusoidal areas. Transmission electron micrographs of livers from animals with fibrosis and treated with genistein showed a reduction in the number of hepatic stellate cells activated and a smaller number of collagen fibers. Genistein is able to improve the liver after injury and fibrosis induced by chronic administration of carbon tetrachloride. This finding suggests that genistein has antifibrogenic potential and could therefore be useful for treating chronic liver disease. (c) 2008 S. Karger AG, Basel.

The tissue-type plasminogen activator–plasminogen activator inhibitor 1 complex promotes neurovascular injury in brain trauma: evidence from mice and humans

PubMed Central

Sashindranath, Maithili; Sales, Eunice; Daglas, Maria; Freeman, Roxann; Samson, Andre L.; Cops, Elisa J.; Beckham, Simone; Galle, Adam; McLean, Catriona; Morganti-Kossmann, Cristina; Rosenfeld, Jeffrey V.; Madani, Rime; Vassalli, Jean-Dominique; Su, Enming J.; Lawrence, Daniel A.

2012-01-01

The neurovascular unit provides a dynamic interface between the circulation and central nervous system. Disruption of neurovascular integrity occurs in numerous brain pathologies including neurotrauma and ischaemic stroke. Tissue plasminogen activator is a serine protease that converts plasminogen to plasmin, a protease that dissolves blood clots. Besides its role in fibrinolysis, tissue plasminogen activator is abundantly expressed in the brain where it mediates extracellular proteolysis. However, proteolytically active tissue plasminogen activator also promotes neurovascular disruption after ischaemic stroke; the molecular mechanisms of this process are still unclear. Tissue plasminogen activator is naturally inhibited by serine protease inhibitors (serpins): plasminogen activator inhibitor-1, neuroserpin or protease nexin-1 that results in the formation of serpin:protease complexes. Proteases and serpin:protease complexes are cleared through high-affinity binding to low-density lipoprotein receptors, but their binding to these receptors can also transmit extracellular signals across the plasma membrane. The matrix metalloproteinases are the second major proteolytic system in the mammalian brain, and like tissue plasminogen activators are pivotal to neurological function but can also degrade structures of the neurovascular unit after injury. Herein, we show that tissue plasminogen activator potentiates neurovascular damage in a dose-dependent manner in a mouse model of neurotrauma. Surprisingly, inhibition of activity following administration of plasminogen activator inhibitor-1 significantly increased cerebrovascular permeability. This led to our finding that formation of complexes between tissue plasminogen activator and plasminogen activator inhibitor-1 in the brain parenchyma facilitates post-traumatic cerebrovascular damage. We demonstrate that following trauma, the complex binds to low-density lipoprotein receptors, triggering the induction of matrix metalloproteinase-3. Accordingly, pharmacological inhibition of matrix metalloproteinase-3 attenuates neurovascular permeability and improves neurological function in injured mice. Our results are clinically relevant, because concentrations of tissue plasminogen activator: plasminogen activator inhibitor-1 complex and matrix metalloproteinase-3 are significantly elevated in cerebrospinal fluid of trauma patients and correlate with neurological outcome. In a separate study, we found that matrix metalloproteinase-3 and albumin, a marker of cerebrovascular damage, were significantly increased in brain tissue of patients with neurotrauma. Perturbation of neurovascular homeostasis causing oedema, inflammation and cell death is an important cause of acute and long-term neurological dysfunction after trauma. A role for the tissue plasminogen activator–matrix metalloproteinase axis in promoting neurovascular disruption after neurotrauma has not been described thus far. Targeting tissue plasminogen activator: plasminogen activator inhibitor-1 complex signalling or downstream matrix metalloproteinase-3 induction may provide viable therapeutic strategies to reduce cerebrovascular permeability after neurotrauma. PMID:22822039

Acetic acid in aged vinegar affects molecular targets for thrombus disease management.

PubMed

Jing, Li; Yanyan, Zhang; Junfeng, Fan

2015-08-01

To elucidate the mechanism underlying the action of dietary vinegar on antithrombotic activity, acetic acid, the main acidic component of dietary vinegar, was used to determine antiplatelet and fibrinolytic activity. The results revealed that acetic acid significantly inhibits adenosine diphosphate (ADP)-, collagen-, thrombin-, and arachidonic acid (AA)-induced platelet aggregation. Acetic acid (2.00 mM) reduced AA-induced platelet aggregation to approximately 36.82 ± 1.31%, and vinegar (0.12 mL L(-1)) reduced the platelet aggregation induced by AA to 30.25 ± 1.34%. Further studies revealed that acetic acid exerts its effects by inhibiting cyclooxygenase-1 and the formation of thromboxane-A2. Organic acids including acetic acid, formic acid, lactic acid, citric acid, and malic acid also showed fibrinolytic activity; specifically, the fibrinolytic activity of acetic acid amounted to 1.866 IU urokinase per mL. Acetic acid exerted its fibrinolytic activity by activating plasminogen during fibrin crossing, thus leading to crosslinked fibrin degradation by the activated plasmin. These results suggest that organic acids in dietary vinegar play important roles in the prevention and cure of cardiovascular diseases.

Fibrinogen catabolism within the procoagulant VX-2 tumor of rabbit lung in vivo: Effluxing fibrin(ogen) fragments contain antiangiogenic activity.

PubMed

Hatton, Mark W C; Southward, Suzanne M R; Legault, Kimberly J; Ross, Bonnie L; Clarke, Bryan J; Bajzar, Laszlo; Blajchman, Morris A; Singh, Gurmit; Richardson, Mary

2004-04-01

Many types of solid tumors are known to be procoagulant environments. This is partly because a hyperpermeable vascular system within the tumor allows plasma hemostatic factors to accumulate in relatively high concentrations in the stroma, and many solid-tumor cells express tissue factor or a procoagulant factor. These circumstances appear to exist in the VX-2 lung tumor of the New Zealand White (NZW) rabbit, and they sustain a measurable turnover of stromal deposits of fibrin(ogen). We have measured the turnover of fibrinogen within tumors of the VX-2 tumor-burdened rabbit and analysed the catabolic products of fibrin(ogen) and the status of fibrinolysis in tumor-derived interpleural effusate. Using intravenously injected (125)I-labeled rabbit fibrinogen as a marker, we found that fibrinogen (approximate blood concentration 1740 microg/mL) passed from blood to VX-2 tumor stroma, saturating the tumor at a concentration of approximately 348 microg fibrinogen/g in approximately 12 hours. We measured fibrin(ogen) fragments, at a concentration of approximately 292 microg/mL, in interpleural effusates that we recovered from 13% of the VX-2-burdened rabbits. Unreduced fibrin(ogen) fragments consisted of 4 major components with a relative molecular mass of approximately 250,000 (assumed to be fragment X; approximately 9% of total fragments from densitometry of immunoblots), 200,000 (d-dimer; 41%), 110,000 (fragment D; 49%), and 50,000 to 55,000 (fragment E; 1%-2%) kD. Total fibrin(ogen) fragments immunopurified from effusates exhibited an antiangiogenic effect when subjected to a chick embryo chorioallantoic membrane procedure. Interpleural effusates were devoid of plasmin activity or active plasminogen activator inhibitor-1 but contained plasmin complexes and active urokinase-like plasminogen activator (uPA), alpha(2)-antiplasmin, and thrombin-activatable fibrinolysis inhibitor. We speculate that VX-2 cells release uPA to activate fibrinolysis within the tumor stroma. Catabolic products of hemostasis (eg, fibrinolytic fragments, angiostatin) flux from the stroma into the interpleural space, thereby providing a net antiangiogenic property to the effusate and ultimately to the lymphatic and circulatory systems.

Effects of losartan on hepatic expression of nonphagocytic NADPH oxidase and fibrogenic genes in patients with chronic hepatitis C.

PubMed

Colmenero, Jordi; Bataller, Ramón; Sancho-Bru, Pau; Domínguez, Marlene; Moreno, Montserrat; Forns, Xavier; Bruguera, Miquel; Arroyo, Vicente; Brenner, David A; Ginès, Pere

2009-10-01

Angiotensin II promotes liver fibrogenesis by stimulating nonphagocytic NADPH oxidase (NOX)-induced oxidative stress. Angiotensin II type 1 (AT1) receptor blockers attenuate experimental liver fibrosis, yet their effects in human liver fibrosis are unknown. We investigated the effects of losartan on hepatic expression of fibrogenic, inflammatory, and NOX genes in patients with chronic hepatitis C (CHC). Fourteen patients with CHC and liver fibrosis received oral losartan (50 mg/day) for 18 mo. Liver biopsies were performed at baseline and after treatment. The degree of inflammation and fibrosis was evaluated by histological analysis (METAVIR). Collagen content was measured by morphometric quantification of Sirius red staining. Overall collagen content and fibrosis stage remained stable in the whole series, yet the fibrosis stage decreased in seven patients. Inflammatory activity improved in seven patients. The effect of losartan on hepatic expression of 31 profibrogenic and inflammatory genes and components of the NOX complex was assessed by quantitative PCR. Losartan treatment was associated with a significant decrease in the expression of several profibrogenic and NOX genes including procollagen alpha1(I) and alpha1(IV), urokinase-type plasminogen activator, metalloproteinase type 2, NOX activator 1 (NOXA-1) and organizer 1 (NOXO-1), and Rac-1. Losartan was well tolerated in all patients and was effective in attenuating the activity of the systemic renin-angiotensin system. No effects on serum liver tests or viral load were observed. We conclude that prolonged administration of losartan, an oral AT1 receptor blocker, is associated with downregulation of NOX components and fibrogenic genes in patients with CHC. Controlled studies are warranted to assess the effect of AT1 receptor blockers in chronic liver injury.

Germinated Brown Rice Attenuates Atherosclerosis and Vascular Inflammation in Low-Density Lipoprotein Receptor-Knockout Mice.

PubMed

Zhao, Ruozhi; Ghazzawi, Nora; Wu, Jiansu; Le, Khuong; Li, Chunyang; Moghadasian, Mohammed H; Siow, Yaw L; Apea-Bah, Franklin B; Beta, Trust; Yin, Zhengfeng; Shen, Garry X

2018-05-02

The present study investigates the impact of germinated brown rice (GBR) on atherosclerosis and the underlying mechanism in low-density lipoprotein receptor-knockout (LDLr-KO) mice. The intensity of atherosclerosis in aortas of LDLr-KO mice receiving diet supplemented with 60% GBR (weight/weight) was significantly less than that in mice fed with 60% white rice (WR) or control diet ( p < 0.05); all diets contained 0.06% cholesterol. WR or GBR diet did not significantly alter plasma total or LDL-cholesterol, fecal sterols, or glucose, or the activities of antioxidant enzymes, compared to the control diet. The adhesion of monocytes to aortas from LDLr-KO mice fed with WR diet was significantly more than that from mice receiving the control diet ( p < 0.01). GBR diet decreased monocyte adhesion to aortas compared to WR diet ( p < 0.01). GBR diet also reduced the levels of plasminogen activator inhibitor-1 (PAI-1), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) in plasma, and the abundances of MCP-1, PAI-1, TNF-α, intracellular cell adhesion molecule-1, toll-like receptor-4, PAI-1, LDLr-like protein, and urokinase plasminogen activator and its receptor in aortas or hearts from LDLr-KO mice in comparison to the WR diet ( p < 0.05, 0.01, respectively). The findings suggest that GBR administration attenuated atherosclerosis and vascular inflammation in LDLr-KO mice compared to WR. The anti-atherosclerotic effect of GBR in LDLr-KO mice at least in part results from its anti-inflammatory activity.

Melanoma cell therapy: Endothelial progenitor cells as shuttle of the MMP12 uPAR-degrading enzyme

PubMed Central

Laurenzana, Anna; Biagioni, Alessio; D'Alessio, Silvia; Bianchini, Francesca; Chillà, Anastasia; Margheri, Francesca; Luciani, Cristina; Mazzanti, Benedetta; Pimpinelli, Nicola; Torre, Eugenio; Danese, Silvio; Calorini, Lido; Rosso, Mario Del; Fibbi, Gabriella

2014-01-01

The receptor for the urokinase-type plasminogen activator (uPAR) accounts for many features of cancer progression, and is therefore considered a target for anti-tumoral therapy. Only full length uPAR mediates tumor progression. Matrix-metallo-proteinase-12 (MMP12)-dependent uPAR cleavage results into the loss of invasion properties and angiogenesis. MMP12 can be employed in the field of “targeted therapies” as a biological drug to be delivered directly in patient's tumor mass. Endothelial Progenitor Cells (EPCs) are selectively recruited within the tumor and could be used as cellular vehicles for delivering anti-cancer molecules. The aim of our study is to inhibit cancer progression by engeneering ECFCs, a subset of EPC, with a lentivirus encoding the anti-tumor uPAR-degrading enzyme MMP12. Ex vivo manipulated ECFCs lost the capacity to perform capillary morphogenesis and acquired the anti-tumor and anti-angiogenetic activity. In vivo MMP12-engineered ECFCs cleaved uPAR within the tumor mass and strongly inhibited tumor growth, tumor angiogenesis and development of lung metastasis. The possibility to exploit tumor homing and activity of autologous MMP12-engineered ECFCs represents a novel way to combat melanoma by a “personalized therapy”, without rejection risk. The i.v. injection of radiolabelled MMP12-ECFCs can thus provide a new theranostic approach to control melanoma progression and metastasis. PMID:25003596

An ELISA method detecting the active form of suPAR.

PubMed

Zhou, Xiaolei; Xu, Mingming; Huang, Hailong; Mazar, Andrew; Iqbal, Zafar; Yuan, Cai; Huang, Mingdong

2016-11-01

Urokinase plasminogen activator receptor (uPAR) exists in a number of formats in human plasma, including soluble uPAR (suPAR) and uPAR fragments. We developed an ELISA method to detect specifically the active form suPAR, which binds to its natural ligand uPA. The intra CV and inter CV of this ELISA assay is 8.5% and 9.6% respectively, and the assay can recover 99.74% of added recombinant suPAR from 10% plasma. This assay is quite sensitive, capable of detecting down to 15pg/ml of suPAR, and can measure suPAR concentrations in the range of 0.031-8ng/ml with high linear relationship. Plasma samples from pregnant women were also measured for the active form of suPAR with this assay, giving an averaged level of 1.39ng/ml, slightly higher than the level of pooled plasma from healthy donors (0.96ng/ml). This study demonstrates the feasibility to measure the active form of suPAR, which will likely have value in clinical applications. Copyright © 2016. Published by Elsevier B.V.

Cardiac macrophages adopt profibrotic/M2 phenotype in infarcted hearts: Role of urokinase plasminogen activator.

PubMed

Carlson, Signe; Helterline, Deri; Asbe, Laura; Dupras, Sarah; Minami, Elina; Farris, Stephen; Stempien-Otero, April

2017-07-01

Macrophages (mac) that over-express urokinase plasminogen activator (uPA) adopt a profibrotic M2 phenotype in the heart in association with cardiac fibrosis. We tested the hypothesis that cardiac macs are M2 polarized in infarcted mouse and human hearts and that polarization is dependent on mac-derived uPA. Studies were performed using uninjured (UI) or infarcted (MI) hearts of uPA overexpressing (SR-uPA), uPA null, or nontransgenic littermate (Ntg) mice. At 7days post-infarction, cardiac mac were isolated, RNA extracted and M2 markers Arg1, YM1, and Fizz1 measured with qrtPCR. Histologic analysis for cardiac fibrosis, mac and myofibroblasts was performed at the same time-point. Cardiac macs were also isolated from Ntg hearts and RNA collected after primary isolation or culture with vehicle, IL-4 or plasmin and M2 marker expression measured. Cardiac tissue and blood was collected from humans with ischemic heart disease. Expression of M2 marker CD206 and M1 marker TNFalpha was measured. Macs from WT mice had increased expression of Arg1 and Ym1 following MI (41.3±6.5 and 70.3±36, fold change vs UI, n=8, P<0.007). There was significant up-regulation of cardiac mac Arg1 and YM1 with MI in both WT and uPA null mice (n=4-9 per genotype and condition). Treatment with plasmin increased expression of Arg1 and YM1 in cultured cardiac macs. Histologic analysis revealed increased density of activated fibroblasts and M2 macs in SR-uPA hearts post-infarction with associated increases in fibrosis. Cardiac macs isolated from human hearts with ischemic heart disease expressed increased levels of the M2 marker CD206 in comparison to blood-derived macs (4.9±1.3). Cardiac macs in mouse and human hearts adopt a M2 phenotype in association with fibrosis. Plasmin can induce an M2 phenotype in cardiac macs. However, M2 activation can occur in the heart in vivo in the absence of uPA indicating that alternative pathways to activate plasmin are present in the heart. Excess uPA promotes increased fibroblast density potentially via potentiating fibroblast migration or proliferation. Altering macrophage phenotype in the heart is a potential target to modify cardiac fibrosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Amine-functionalized lanthanide-doped zirconia nanoparticles: optical spectroscopy, time-resolved fluorescence resonance energy transfer biodetection, and targeted imaging.

PubMed

Liu, Yongsheng; Zhou, Shanyong; Tu, Datao; Chen, Zhuo; Huang, Mingdong; Zhu, Haomiao; Ma, En; Chen, Xueyuan

2012-09-12

Ultrasmall inorganic oxide nanoparticles doped with trivalent lanthanide ions (Ln(3+)), a new and huge family of luminescent bioprobes, remain nearly untouched. Currently it is a challenge to synthesize biocompatible ultrasmall oxide bioprobes. Herein, we report a new inorganic oxide bioprobe based on sub-5 nm amine-functionalized tetragonal ZrO(2)-Ln(3+) nanoparticles synthesized via a facile solvothermal method and ligand exchange. By utilizing the long-lived luminescence of Ln(3+), we demonstrate its application as a sensitive time-resolved fluorescence resonance energy transfer (FRET) bioprobe to detect avidin with a record-low detection limit of 3.0 nM. The oxide nanoparticles also exhibit specific recognition of cancer cells overexpressed with urokinase plasminogen activator receptor (uPAR, an important marker of tumor biology and metastasis) and thus may have great potentials in targeted bioimaging.

Quebec platelet disorder: update on pathogenesis, diagnosis, and treatment.

PubMed

Blavignac, Jessica; Bunimov, Natalia; Rivard, Georges E; Hayward, Catherine P M

2011-09-01

Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder associated with reduced platelet counts and a unique gain-of-function defect in fibrinolysis due to increased expression and storage of urokinase plasminogen activator (uPA) by megakaryocytes. QPD increases risks for bleeding and its key clinical feature is delayed-onset bleeding, following surgery, dental procedures or trauma, which responds only to treatment with fibrinolytic inhibitors. The genetic cause of the disorder is a tandem duplication mutation of the uPA gene, PLAU, which upregulates uPA expression in megakaryocytes by an unknown mechanism. The increased platelet stores of uPA trigger plasmin-mediated degradation of QPD α-granule proteins. The gain-of-function defect in fibrinolysis is thought to be central to the pathogenesis of QPD bleeding as the activation of QPD platelets leads to release of uPA from α-granules and accelerated clot lysis. The purpose of this review is to summarize current knowledge on QPD pathogenesis and the recommended approaches to QPD diagnosis and treatment. Thieme Medical Publishers.

Effects of Lewis lung carcinoma on trabecular microstructural changes in wild-type and plasminogen activator inhibitor-1 deficient mice fed a high-fat diet

USDA-ARS?s Scientific Manuscript database

Bone is a major target organ of metastasis. The present study investigated the effects of Lewis lung carcinoma (LLC) on trabecular microstructural changes, using tomographic analysis, in distal femur and lumbar 4 vertebra from LLC-bearing wild-type and plasminogen activator inhibitor-1 (PAI-1) defi...

Fiber intake and plasminogen activator inhibitor-1 in type 2 diabetes: Look AHEAD (Action for Health in Diabetes) Trial findings at baseline and 1 year

USDA-ARS?s Scientific Manuscript database

Plasminogen activator inhibitor 1 (PAI-1) is elevated in obese individuals with type 2 diabetes and may contribute, independently of traditional factors, to increased cardiovascular disease risk. Fiber intake may decrease PAI-1 levels. We examined the associations of fiber intake and its changes wit...

Current status of prediction of drug disposition and toxicity in humans using chimeric mice with humanized liver.

PubMed

Kitamura, Shigeyuki; Sugihara, Kazumi

2014-01-01

1. Human-chimeric mice with humanized liver have been constructed by transplantation of human hepatocytes into several types of mice having genetic modifications that injure endogenous liver cells. Here, we focus on liver urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice, which are the most widely used human-chimeric mice. Studies so far indicate that drug metabolism, drug transport, pharmacological effects and toxicological action in these mice are broadly similar to those in humans. 2. Expression of various drug-metabolizing enzymes is known to be different between humans and rodents. However, the expression pattern of cytochrome P450, aldehyde oxidase and phase II enzymes in the liver of human-chimeric mice resembles that in humans, not that in the host mice. 3. Metabolism of various drugs, including S-warfarin, zaleplon, ibuprofen, naproxen, coumarin, troglitazone and midazolam, in human-chimeric mice is mediated by human drug-metabolizing enzymes, not by host mouse enzymes, and thus resembles that in humans. 4. Pharmacological and toxicological effects of various drugs in human-chimeric mice are also similar to those in humans. 5. The current consensus is that chimeric mice with humanized liver are useful to predict drug metabolism catalyzed by cytochrome P450, aldehyde oxidase and phase II enzymes in humans in vivo and in vitro. Some remaining issues are discussed in this review.

Enhanced tissue factor pathway activity and fibrin turnover in the alveolar compartment of patients with interstitial lung disease.

PubMed

Günther, A; Mosavi, P; Ruppert, C; Heinemann, S; Temmesfeld, B; Velcovsky, H G; Morr, H; Grimminger, F; Walmrath, D; Seeger, W

2000-06-01

Bronchoalveolar lavage fluids (BALF) from patients with hypersensitivity pneumonitis (HP; n = 35), idiopathic pulmonary fibrosis (IPF, n = 41) and sarcoidosis (SARC, n = 48) were investigated for alterations in the alveolar hemostatic balance. Healthy individuals (n = 21) served as Controls. Procoagulant activity (PCA), tissue factor (TF) activity and F VII activity were assessed by means of specific recalcification assays. The overall fibrinolytic activity (FA) was measured using the (125)I-labeled fibrin plate assay. Fibrinopeptide A (FP-A), D-Dimer, plasminogen activators (PA) of the urokinase (u-PA) or tissue type (t-PA), PA-inhibitor I (PAI-1) and alpha2-antiplasmin (alpha2-AP) were determined by ELISA technique. As compared to Controls, all groups with interstitial lung disease (ILD) displayed an increase in BALF PCA by approximately one order of magnitude, and this was ascribed to enhanced TF activity by >98%. Accordingly, F VII-activity was increased in all ILD groups, and elevated FP-A levels were noted. There was no significant difference in procoagulant activities between the different ILD entities, but the increase in TF was significantly correlated with deterioration of lung compliance. Overall fibrinolytic activity did not significantly differ between ILD entities and Controls, although some reduction in IPF subjects was observed. Nevertheless, changes in the profile of the different pro- and antifibrinolytic compounds were noted. U-PA, but not t-PA levels were significantly reduced in all ILD groups. alpha2-AP was markedly elevated throughout, whereas PAI-1 levels were lowered. As a balance of

A Novel Functional Role of Collagen Glycosylation

PubMed Central

Jürgensen, Henrik J.; Madsen, Daniel H.; Ingvarsen, Signe; Melander, Maria C.; Gårdsvoll, Henrik; Patthy, Laszlo; Engelholm, Lars H.; Behrendt, Niels

2011-01-01

Collagens make up the most abundant component of interstitial extracellular matrices and basement membranes. Collagen remodeling is a crucial process in many normal physiological events and in several pathological conditions. Some collagen subtypes contain specific carbohydrate side chains, the function of which is poorly known. The endocytic collagen receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 plays an important role in matrix remodeling through its ability to internalize collagen for lysosomal degradation. uPARAP/Endo180 is a member of the mannose receptor protein family. These proteins all include a fibronectin type II domain and a series of C-type lectin-like domains, of which only a minor part possess carbohydrate recognition activity. At least two of the family members, uPARAP/Endo180 and the mannose receptor, interact with collagens. The molecular basis for this interaction is known to involve the fibronectin type II domain but nothing is known about the function of the lectin domains in this respect. In this study, we have investigated a possible role of the single active lectin domain of uPARAP/Endo180 in the interaction with collagens. By expressing truncated recombinant uPARAP/Endo180 proteins and analyzing their interaction with collagens with high and low levels of glycosylation we demonstrated that this lectin domain interacts directly with glycosylated collagens. This interaction is functionally important because it was found to modulate the endocytic efficiency of the receptor toward highly glycosylated collagens such as basement membrane collagen IV. Surprisingly, this property was not shared by the mannose receptor, which internalized glycosylated collagens independently of its lectin function. This role of modulating its uptake efficiency by a specific receptor is a previously unrecognized function of collagen glycosylation. PMID:21768090

The antiprotease SPINK7 serves as an inhibitory checkpoint for esophageal epithelial inflammatory responses.

PubMed

Azouz, Nurit P; Ynga-Durand, Mario A; Caldwell, Julie M; Jain, Ayushi; Rochman, Mark; Fischesser, Demetria M; Ray, Leanne M; Bedard, Mary C; Mingler, Melissa K; Forney, Carmy; Eilerman, Matthew; Kuhl, Jonathan T; He, Hua; Biagini Myers, Jocelyn M; Mukkada, Vincent A; Putnam, Philip E; Khurana Hershey, Gurjit K; Kottyan, Leah C; Wen, Ting; Martin, Lisa J; Rothenberg, Marc E

2018-06-06

Loss of barrier integrity has an important role in eliciting type 2 immune responses, yet the molecular events that initiate and connect this with allergic inflammation remain unclear. We reveal an endogenous, homeostatic mechanism that controls barrier function and inflammatory responses in esophageal allergic inflammation. We show that a serine protease inhibitor, SPINK7 (serine peptidase inhibitor, kazal type 7), is part of the differentiation program of human esophageal epithelium and that SPINK7 depletion occurs in a human allergic, esophageal condition termed eosinophilic esophagitis. Experimental manipulation strategies reducing SPINK7 in an esophageal epithelial progenitor cell line and primary esophageal epithelial cells were sufficient to induce barrier dysfunction and transcriptional changes characterized by loss of cellular differentiation and altered gene expression known to stimulate allergic responses (for example, FLG and SPINK5 ). Epithelial silencing of SPINK7 promoted production of proinflammatory cytokines including thymic stromal lymphopoietin (TSLP). Loss of SPINK7 increased the activity of urokinase plasminogen-type activator (uPA), which in turn had the capacity to promote uPA receptor-dependent eosinophil activation. Treatment of epithelial cells with the broad-spectrum antiserine protease, α1 antitrypsin, reversed the pathologic features associated with SPINK7 silencing. The relevance of this pathway in vivo was supported by finding genetic epistasis between variants in TSLP and the uPA-encoding gene, PLAU We propose that the endogenous balance between SPINK7 and its target proteases is a key checkpoint in regulating mucosal differentiation, barrier function, and inflammatory responses and that protein replacement with antiproteases may be therapeutic for select allergic diseases. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Vitamin D binding protein-macrophage activating factor directly inhibits proliferation, migration, and uPAR expression of prostate cancer cells.

PubMed

Gregory, Kalvin J; Zhao, Bing; Bielenberg, Diane R; Dridi, Sami; Wu, Jason; Jiang, Weihua; Huang, Bin; Pirie-Shepherd, Steven; Fannon, Michael

2010-10-18

Vitamin D binding protein-macrophage activating factor (DBP-maf) is a potent inhibitor of tumor growth. Its activity, however, has been attributed to indirect mechanisms such as boosting the immune response by activating macrophages and inhibiting the blood vessel growth necessary for the growth of tumors. In this study we show for the first time that DBP-maf exhibits a direct and potent effect on prostate tumor cells in the absence of macrophages. DBP-maf demonstrated inhibitory activity in proliferation studies of both LNCaP and PC3 prostate cancer cell lines as well as metastatic clones of these cells. Flow cytometry studies with annexin V and propidium iodide showed that this inhibitory activity is not due to apoptosis or cell death. DBP-maf also had the ability to inhibit migration of prostate cancer cells in vitro. Finally, DBP-maf was shown to cause a reduction in urokinase plasminogen activator receptor (uPAR) expression in prostate tumor cells. There is evidence that activation of this receptor correlates with tumor metastasis. These studies show strong inhibitory activity of DBP-maf on prostate tumor cells independent of its macrophage activation.

Vitamin D Binding Protein-Macrophage Activating Factor Directly Inhibits Proliferation, Migration, and uPAR Expression of Prostate Cancer Cells

PubMed Central

Bielenberg, Diane R.; Dridi, Sami; Wu, Jason; Jiang, Weihua; Huang, Bin; Pirie-Shepherd, Steven; Fannon, Michael

2010-01-01

Background Vitamin D binding protein-macrophage activating factor (DBP-maf) is a potent inhibitor of tumor growth. Its activity, however, has been attributed to indirect mechanisms such as boosting the immune response by activating macrophages and inhibiting the blood vessel growth necessary for the growth of tumors. Methods and Findings In this study we show for the first time that DBP-maf exhibits a direct and potent effect on prostate tumor cells in the absence of macrophages. DBP-maf demonstrated inhibitory activity in proliferation studies of both LNCaP and PC3 prostate cancer cell lines as well as metastatic clones of these cells. Flow cytometry studies with annexin V and propidium iodide showed that this inhibitory activity is not due to apoptosis or cell death. DBP-maf also had the ability to inhibit migration of prostate cancer cells in vitro. Finally, DBP-maf was shown to cause a reduction in urokinase plasminogen activator receptor (uPAR) expression in prostate tumor cells. There is evidence that activation of this receptor correlates with tumor metastasis. Conclusions These studies show strong inhibitory activity of DBP-maf on prostate tumor cells independent of its macrophage activation. PMID:20976141

Involvement of nitric oxide synthase in matrix metalloproteinase-9- and/or urokinase plasminogen activator receptor-mediated glioma cell migration

PubMed Central

2013-01-01

Background Src tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Our recent studies have clearly demonstrated the role of α9β1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we evaluated the involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration. Methods MMP-9 and uPAR shRNAs and overexpressing plasmids were used to downregulate and upregulate these molecules, respectively in U251 glioma cells and 5310 glioma xenograft cells. The effect of treatments on migration and invasion potential of these glioma cells were assessed by spheroid migration, wound healing, and Matrigel invasion assays. In order to attain the other objectives we also performed immunocytochemical, immunohistochemical, RT-PCR, Western blot and fluorescence-activated cell sorting (FACS) analysis. Results Immunohistochemical analysis revealed the prominent association of iNOS with glioblastoma multiforme (GBM). Immunofluorescence analysis showed prominent expression of iNOS in glioma cells. MMP-9 and/or uPAR knockdown by respective shRNAs reduced iNOS expression in these glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in either MMP-9 or uPAR overexpressed glioma cells. The migration potential of MMP-9- and/or uPAR-overexpressed U251 glioma cells was significantly inhibited after treatment with L-NAME, an inhibitor of iNOS. Similarly, a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed glioma cells was noticed after L-NAME treatment. A prominent reduction of iNOS expression was observed in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Protein expressions of cSrc, phosphoSrc and p130Cas were reduced with simultaneous knockdown of both MMP-9 and uPAR. Conclusions Taken together, our results from the present and earlier studies clearly demonstrate that α9β1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced α9β1 integrin and iNOS levels. PMID:24325546

Domain retention in transcription factor fusion genes and its biological and clinical implications: a pan-cancer study

PubMed Central

Kim, Pora; Ballester, Leomar Y.; Zhao, Zhongming

2017-01-01

Genomic rearrangements involving transcription factors (TFs) can form fusion proteins resulting in either enhanced, weakened, or even loss of TF activity. Functional domain (FD) retention is a critical factor in the activity of transcription factor fusion genes (TFFGs). A systematic investigation of FD retention in TFFGs and their outcome (e.g. expression changes) in a pan-cancer study has not yet been completed. Here, we examined the FD retention status in 386 TFFGs across 13 major cancer types and identified 83 TFFGs involving 67 TFs that retained FDs. To measure the potential biological relevance of TFs in TFFGs, we introduced a Major Active Isofusion Index (MAII) and built a prioritized TFFG network using MAII scores and the observed frequency of fusion positive samples. Interestingly, the four TFFGs (PML-RARA, RUNX1-RUNX1T1, TMPRSS2-ERG, and SFPQ-TFE3) with the highest MAII scores showed 50 differentially expressed target genes (DETGs) in fusion-positive versus fusion-negative cancer samples. DETG analysis revealed that they were involved in tumorigenesis-related processes in each cancer type. PLAU, which encodes plasminogen activator urokinase and serves as a biomarker for tumor invasion, was found to be consistently activated in the samples with the highest MAII scores. Among the 50 DETGs, 21 were drug targetable genes. Fourteen of these 21 DETGs were expressed in acute myeloid leukemia (AML) samples. Accordingly, we constructed an AML-specific TFFG network, which included 38 DETGs in RUNX1-RUNX1T1 or PML-RARA positive samples. In summary, this study revealed several TFFGs and their potential target genes, and provided insights into the clinical implications of TFFGs. PMID:29299133

Regulation of HGF and c-MET Interaction in Normal Ovary and Ovarian Cancer.

PubMed

Kwon, Youngjoo; Godwin, Andrew K

2017-04-01

Binding of hepatocyte growth factor (HGF) to the c-MET receptor has mitogenic, motogenic, and morphogenic effects on cells. The versatile biological effects of HGF and c-MET interactions make them important contributors to the development of malignant tumors. We and others have demonstrated a therapeutic value in targeting the interaction of c-MET and HGF in epithelial ovarian cancer (EOC). However, both HGF and c-MET are expressed in the normal ovary as well. Therefore, it is important to understand the differences in mechanisms that control HGF signaling activation and its functional role in the normal ovary and EOC. In the normal ovary, HGF signaling may be under hormonal regulation. During ovulation, HGF-converting proteases are secreted and the subsequent activation of HGF signaling enhances the proliferation of ovarian surface epithelium in order to replenish the area damaged due to expulsion of the ovum. In contrast, EOC cells that exhibit epithelial characteristics constitutively express both c-MET and HGF-converting proteases such as urokinase-type plasminogen activator. In EOC, mechanisms to control the activation of HGF signaling are absent since HGF is provided locally from the tissue microenvironment as well as remotely throughout the body. Potential incessant HGF signaling in EOC may lead to an increase in proliferation, invasion through the stroma, and migration to other tissues of cancer cells. Therefore, targeting the interaction of c-MET and HGF would be beneficial in treating EOC.

Regulation of HGF and c-MET Interaction in Normal Ovary and Ovarian Cancer

PubMed Central

Kwon, Youngjoo; Godwin, Andrew K.

2016-01-01

Binding of hepatocyte growth factor (HGF) to the c-MET receptor has mitogenic, motogenic, and morphogenic effects on cells. The versatile biological effects of HGF and c-MET interactions make them important contributors to the development of malignant tumors. We and others have demonstrated a therapeutic value in targeting the interaction of c-MET and HGF in epithelial ovarian cancer (EOC). However, both HGF and c-MET are expressed in the normal ovary as well. Therefore, it is important to understand the differences in mechanisms that control HGF signaling activation and its functional role in the normal ovary and EOC. In the normal ovary, HGF signaling may be under hormonal regulation. During ovulation, HGF-converting proteases are secreted and the subsequent activation of HGF signaling enhances the proliferation of ovarian surface epithelium in order to replenish the area damaged due to expulsion of the ovum. In contrast, EOC cells that exhibit epithelial characteristics constitutively express both c-MET and HGF-converting proteases such as urokinase-type plasminogen activator. In EOC, mechanisms to control the activation of HGF signaling are absent since HGF is provided locally from the tissue microenvironment as well as remotely throughout the body. Potential incessant HGF signaling in EOC may lead to an increase in proliferation, invasion through the stroma, and migration to other tissues of cancer cells. Therefore, targeting the interaction of c-MET and HGF would be beneficial in treating EOC. PMID:27170665

Alterations of fibrin network structure mediated by dermatan sulfate.

PubMed

Lauricella, Ana María; Castañon, María Mercedes; Kordich, Lucía C; Quintana, Irene L

2013-02-01

Dermatan sulfate (DS) is well-known for its anticoagulant activity through binding to heparin cofactor II (HCII) to enhance thrombin inhibition. It has also been reported that DS has a profibrinolytic effect. We have evaluated the effects of DS solutions (4-20 μg/mL) on the formation (by kinetic studies), structure (by electron microscopy and compaction assays) and lysis (with urokinase-type plasminogen activator) of plasma fibrin networks. The results showed that DS significantly prolonged the lag phase and decreased the fibrin formation rate and the optical density of the final networks versus control, in a concentration dependent way. DS-associated networks presented a minor network percentage compared with control, composed of lower number of fibers per field, which resulted significantly thinner and longer. Moreover, DS rendered gels more sensible to rupture by centrifugal force and more susceptible to lysis. When fibrin formation kinetic assays were performed with purified fibrinogen instead of plasma, in the absence of HCII, the optical density of final DS-associated networks was statistically lower than control. Therefore, a direct effect of DS on the thickness of fibers was observed. Since in all in vitro assays low DS concentrations were used, it could be postulated that the fibrin features described above are plausible to be found in in vivo thrombi and therefore, DS would contribute to the formation of less thrombogenic clots.

Extravascular plasminogen activator and inhibitor activities detected at the site of a chronic mycobacterial-induced inflammation.

PubMed Central

O'Rourke, J.; Wang, W. P.; Donnelly, L.; Wang, E.; Kreutzer, D. L.

1987-01-01

Levels of extravascular tissue plasminogen activator activity (PA) and those of inhibitors of PA and of urokinase (UK) present within the anterior chamber of normal and inflamed feline eyes were assessed with the use of a direct PA assay of microsamples of aqueous humor. Purposes of the study were, first, to confirm prior indirect evidence that this extravascular space normally contains higher levels of uninhibited PA, but lower levels of inhibitor activity, than does plasma and, second, to determine patterns of change in these activities under in vivo conditions imposed by a chronic mycobacterial-induced uveitis (CMIU) disease model. The PA assay utilized a 125I-plasminogen substrate whose cleavage by PA contained in samples was both visualized during gel electrophoreis, and quantified by gamma counting. The results provided the first direct evidence that the higher fibrinolytic activity previously observed in normal aqueous in comparison with plasma is in fact associated with higher levels of available (uninhibited) PA (P less than 0.01) The data also indicated that normal aqueous contains a much higher level of PA inhibitor activity than previously suspected--roughly 40 times more than available PA levels. These normal values for PA and inhibitors occupied a relatively narrow, threefold range, in contrast to the wide scattering of individual values that appeared during 18-20 weeks of the chronic inflammation disease model. Despite this, however, the general pattern of observation for all individual eyes during CMIU was a significant increase in levels of both PA and inhibitors. The net effect of CMIU was thus to cause the 1:40 ratio noted above to be tilted more strongly in favor of inhibitor activity, ie, up to 1:80. Increases in local vasopermeability in this disease model were believed contributory to this change. However, local generations of PA and APA in vivo by inflammatory cells, especially monocyte-macrophages, must also be considered. Assays for UK inhibitor showed levels of activity and directions of change that closely followed those of PA inhibitor, which suggests the possibility that they may be identical. It is surmised that the above patterns, along with results of our prior studies, indicate an apparent need for a multistep, strict inhibitory control of plasmin generation and proteolysis in vivo within normal extravascular spaces such as the anterior chamber.(ABSTRACT TRUNCATED AT 400 WORDS) Images Figure 2 PMID:3493701

Carnosic acid inhibits the proliferation and migration capacity of human colorectal cancer cells

PubMed Central

BARNI, M.V.; CARLINI, M.J.; CAFFERATA, E.G.; PURICELLI, L.; MORENO, S.

2012-01-01

Colorectal cancer (CRC) is the third most common malignant neoplasm worldwide. The objective of this study was to examine whether carnosic acid (CA), the main antioxidant compound of Rosmarinus officinalis L., would inhibit the cell viability of three CRC cell lines: Caco-2, HT29 and LoVo in a dose-dependent manner, with IC50 values in the range of 24–96 μM. CA induced cell death by apoptosis in Caco-2 line after 24 h of treatment and inhibited cell adhesion and migration, possibly by reducing the activity of secreted proteases such as urokinase plasminogen activator (uPA) and metalloproteinases (MMPs). These effects may be associated through a mechanism involving the inhibition of the COX-2 pathway, because we have determined that CA downregulates the expression of COX-2 in Caco-2 cells at both the mRNA and protein levels. Therefore, CA modulates different targets involved in the development of CRC. These findings indicate that carnosic acid may have anticancer activity and may be useful as a novel chemotherapeutic agent. PMID:22246562

Biochemical assays on plasminogen activators and hormones from kidney sources

NASA Technical Reports Server (NTRS)

Barlow, Grant H.; Lewis, Marian L.; Morrison, Dennis R.

1988-01-01

Investigations were established for the purpose of analyzing the conditioned media from human embryonic kidney cell subpopulations separated in space by electrophoresis. This data is based on the experiments performed on STS-8 on the continuous flow electrophoresis system. The primary biological activity that was analyzed was plasminogen activator activity, but some assays for erythropoeitin and human granulocyte colony stimulating activity were also performed. It is concluded that a battery of assays are required to completely define the plasminogen activator profile of a conditioned media from cell culture. Each type of assay measures different parts of the mixture and are influenced by different parameters. The functional role of each assay is given along with an indication of which combination of assays are required to answer specific questions. With this type of information it is possible by combinations of assays with mathematical analysis to pinpoint a specific component of the system.

5-Benzylglycinyl-Amiloride Kills Proliferating and Nonproliferating Malignant Glioma Cells through Caspase-Independent Necroptosis Mediated by Apoptosis-Inducing Factor

PubMed Central

Pasupuleti, Nagarekha; Leon, Leonardo; Carraway, Kermit L.

2013-01-01

5′–Βenzylglycinyl-amiloride (UCD38B) and glycinyl-amiloride (UCD74A) are cell-permeant and cell-impermeant derivatives of amiloride, respectively, and used here to identify the cellular mechanisms of action underlying their antiglioma effects. UCD38B comparably kills proliferating and nonproliferating gliomas cells when cell cycle progression is arrested either by cyclin D1 siRNA or by acidification. Cell impermeant UCD74A inhibits plasmalemmal urokinase plasminogen activator (uPA) and the type 1 sodium-proton exchanger with potencies analogous to UCD38B, but is cytostatic. In contrast, UCD38B targets intracellular uPA causing mistrafficking of uPA into perinuclear mitochondria, reducing the mitochondrial membrane potential, and followed by the release of apoptotic inducible factor (AIF). AIF nuclear translocation is followed by a caspase-independent necroptotic cell death. Reduction in AIF expression by siRNA reduces the antiglioma cytotoxic effects of UCD38B, while not activating the caspase pathway. Ultrastructural changes shortly following treatment with UCD38B demonstrate dilation of endoplasmic reticulum (ER) and mitochondrial swelling followed by nuclear condensation within hours consistent with a necroptotic cell death differing from apoptosis and from autophagy. These drug mechanism of action studies demonstrate that UCD38B induces a cell cycle-independent, caspase-independent necroptotic glioma cell death that is mediated by AIF and independent of poly (ADP-ribose) polymerase and H2AX activation. PMID:23241369

Severe necroinflammatory reaction caused by natural killer cell-mediated Fas/Fas ligand interaction and dendritic cells in human hepatocyte chimeric mouse.

PubMed

Okazaki, Akihito; Hiraga, Nobuhiko; Imamura, Michio; Hayes, C Nelson; Tsuge, Masataka; Takahashi, Shoichi; Aikata, Hiroshi; Abe, Hiromi; Miki, Daiki; Ochi, Hidenori; Tateno, Chise; Yoshizato, Katsutoshi; Ohdan, Hideki; Chayama, Kazuaki

2012-08-01

The necroinflammatory reaction plays a central role in hepatitis B virus (HBV) elimination. Cluster of differentiation (CD)8-positive cytotoxic T lymphocytes (CTLs) are thought to be a main player in the elimination of infected cells, and a recent report suggests that natural killer (NK) cells also play an important role. Here, we demonstrate the elimination of HBV-infected hepatocytes by NK cells and dendritic cells (DCs) using urokinase-type plasminogen activator/severe combined immunodeficiency mice, in which the livers were highly repopulated with human hepatocytes. After establishing HBV infection, we injected human peripheral blood mononuclear cells (PBMCs) into the mice and analyzed liver pathology and infiltrating human immune cells with flow cytometry. Severe hepatocyte degeneration was observed only in HBV-infected mice transplanted with human PBMCs. We provide the first direct evidence that massive liver cell death can be caused by Fas/Fas ligand (FasL) interaction provided by NK cells activated by DCs. Treatment of mice with anti-Fas antibody completely prevented severe hepatocyte degeneration. Furthermore, severe hepatocyte death can be prevented by depletion of DCs, whereas depletion of CD8-positive CTLs did not disturb the development of massive liver cell apoptosis. Our findings provide the first direct evidence that DC-activated NK cells induce massive HBV-infected hepatocyte degeneration through the Fas/FasL system and may indicate new therapeutic implications for acute severe/fulminant hepatitis B. Copyright © 2012 American Association for the Study of Liver Diseases.

Protein-Binding RNA Aptamers Affect Molecular Interactions Distantly from Their Binding Sites

PubMed Central

Dupont, Daniel M.; Thuesen, Cathrine K.; Bøtkjær, Kenneth A.; Behrens, Manja A.; Dam, Karen; Sørensen, Hans P.; Pedersen, Jan S.; Ploug, Michael; Jensen, Jan K.; Andreasen, Peter A.

2015-01-01

Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site. PMID:25793507

Micro-RNA profile and proteins in peritoneal fluid from women with endometriosis: their relationship with sterility.

PubMed

Marí-Alexandre, Josep; Barceló-Molina, Moisés; Belmonte-López, Elisa; García-Oms, Javier; Estellés, Amparo; Braza-Boïls, Aitana; Gilabert-Estellés, Juan

2018-04-01

To define the microRNA (miRNA) profile and its relationship with cytokines content in peritoneal fluid (PF) from endometriosis patients. Case-control study. University hospital, research institute. One hundred twenty-six women with endometriosis (EPF) and 45 control women (CPF). MiRNA arrays were prepared from six EPF and six CPF. Quantitative reverse transcription-polymerase chain reaction validation of nine selected miRNAs (miR-29c-3p, -106b-3p, -130a-3p, -150-5p, -185-5p, -195-5p, -451a, -486-5p, and -1343-5p) was performed. Vascular endothelial growth factor-A (VEGF-A), thrombospondin-1 (TSP-1), urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), matrix metalloproteinase-3 (MMP3), tissue inhibitor of metalloproteinases type 1 (TIMP-1), interleukin (IL)-6, IL-8, IL-17A, macrophage inflammatory protein 1β (MIP1beta), platelet-derived growth factor α-polypeptide A, and regulated on activation, normal T cell expressed and secreted (RANTES) were quantified by ELISA and MILLIPLEX. MiRNA arrays showed 126 miRNAs differentially expressed (fold change ±1.2) (78 down-regulated, 48 up-regulated) in EPF. Validation showed higher levels of miR-106b-3p, -451a, -486-5p, IL-6, IL-8, uPA, and TIMP-1 in EPF. In menstrual phase, EPF presented up-regulation of miR-106b-3p, -130a-3p, -150-5p, -185-5p, -451a, -486-5p, VEGF-A, IL-8, MIF 1β, uPA, and PAI-1 compared with other phases; however, CPF did not. MiRNA-486-5p was up-regulated in sterile EPF compared with sterile controls, and VEGF-A, IL-8, and TIMP-1 were increased in sterile and fertile EPF compared with fertile CPF. MiRNAs seem to be involved in the peritoneal alterations in endometriosis, suggesting new mechanisms by which ectopic lesions could implant in endometriosis patients; and to serve as biomarkers for fertility outcome prediction. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Effect of dihydrotestosterone on the expression of mucin 1 and the activity of Wnt signaling in mouse corneal epithelial cells.

PubMed

Qin, Li; Pei, Cheng; Kang, Qian-Yan; Liu, Zhao; Li, Li

2016-01-01

To explore the effects of the androgen dihydrotestosterone on the expression of mucin 1 (MUC1) and the activity of Wnt signaling in mouse corneal epithelial cells. Primary mouse corneal epithelial cells were isolated from the corneas of BALB/c mice. Quantitative real-time polymerase chain reaction, immunofluorescence and Western blot analysis were used to quantify the differential expression of selected genes. The androgen receptor was silenced by transfecting cells with androgen receptor shRNAs. TOP-Flash and FOP-flash reporter plasmids were used to measure β-catenin-driven transcription. Dihydrotestosterone treatment increased MUC1 expression and activated the Wnt signaling pathway and led to the translocation of β-catenin and upregulation of the Wnt downstream target gene TATA box binding protein and urokinase plasminogen activator. These effects were prevented by downregulating the androgen receptor. Androgens may protect against dry eye by regulating the expression of MUC1 which is stimulated by the activation of Wnt signaling via the androgen receptor. An understanding of the mechanisms associated with androgen-mediated protection against dry eye is an important step in developing new therapies for this disease.

EMMPRIN/CD147 up-regulates urokinase-type plasminogen activator: implications in oral tumor progression

PubMed Central

2012-01-01

Backgrounds An elevated level of EMMPRIN in cancer tissues have been correlated with tumor invasion in numerous cancers including oral cavity and larynx. Although EMMPRIN's effect has been generally attributed to its MMP inducing activity, we have previously demonstrated in breast cancer model that EMMPRIN can also enhance invasion by upregulating uPA. In this study, the role of EMMPRIN in regulating uPA and invasion was investigated in oral squamous cell carcinoma (OSCC) progression. Methods Precancerous and invasive oral tumoral tissues were used as well as the corresponding cell lines, DOK and SCC-9 respectively. The paracrine regulation of uPA by EMMPRIN was investigated by treating culture cells with EMMPRIN-enriched membrane vesicles. UPA expression was analyzed by qPCR and immunostaining and the consequence on the invasion capacity was studied using modified Boyden chamber assay, in the presence or absence of EMMPRIN blocking antibody, the uPA inhibitor amiloride or the MMP inhibitor marimastat. Results OSCC tumors were shown to express more EMMPRIN and uPA compared to dysplastic lesions. The corresponding cell models, SCC-9 and DOK cells, displayed similar expression pattern. In both cell types EMMPRIN upregulated the expression of uPA as well as that of MMP-2 and MMP-9. EMMPRIN treatment led to a significant increase in cell invasion both in the invasive SCC-9 and in the less invasive dysplastic DOK cells, in an MMP and uPA dependent manner. Conclusions Our results suggest that the upregulation of uPA contributes to EMMPRIN's effect in promoting oral tumor invasion. PMID:22443116

EMMPRIN/CD147 up-regulates urokinase-type plasminogen activator: implications in oral tumor progression.

PubMed

Lescaille, Géraldine; Menashi, Suzanne; Cavelier-Balloy, Bénédicte; Khayati, Farah; Quemener, Cathy; Podgorniak, Marie Pierre; Naïmi, Benyoussef; Calvo, Fabien; Lebbe, Céleste; Mourah, Samia

2012-03-23

An elevated level of EMMPRIN in cancer tissues have been correlated with tumor invasion in numerous cancers including oral cavity and larynx. Although EMMPRIN's effect has been generally attributed to its MMP inducing activity, we have previously demonstrated in breast cancer model that EMMPRIN can also enhance invasion by upregulating uPA. In this study, the role of EMMPRIN in regulating uPA and invasion was investigated in oral squamous cell carcinoma (OSCC) progression. Precancerous and invasive oral tumoral tissues were used as well as the corresponding cell lines, DOK and SCC-9 respectively. The paracrine regulation of uPA by EMMPRIN was investigated by treating culture cells with EMMPRIN-enriched membrane vesicles. UPA expression was analyzed by qPCR and immunostaining and the consequence on the invasion capacity was studied using modified Boyden chamber assay, in the presence or absence of EMMPRIN blocking antibody, the uPA inhibitor amiloride or the MMP inhibitor marimastat. OSCC tumors were shown to express more EMMPRIN and uPA compared to dysplastic lesions. The corresponding cell models, SCC-9 and DOK cells, displayed similar expression pattern. In both cell types EMMPRIN upregulated the expression of uPA as well as that of MMP-2 and MMP-9. EMMPRIN treatment led to a significant increase in cell invasion both in the invasive SCC-9 and in the less invasive dysplastic DOK cells, in an MMP and uPA dependent manner. Our results suggest that the upregulation of uPA contributes to EMMPRIN's effect in promoting oral tumor invasion.

Timing of transcriptomic and proteomic changes in the bovine placentome after parturition.

PubMed

McNeel, Anthony K; Ondrak, Jeff D; Amundson, Olivia L; Fountain, Tara H; Wright, Elane C; Whitman, Katherine J; Chitko-McKown, Carol G; Jones, Shuna A; Chase, Chadwick C; Cushman, Robert A

2017-09-15

Proper post-partum reproductive performance is important for reproductive efficiency in beef cows, and dystocia decreases post-partum fertility. Crossbred beef cows (n = 1676) were evaluated for lifetime performance based on degree of dystocia at presentation of the first calf. Cows that experienced moderate or severe dystocia produced fewer calves during their productive life (P < 0.01). The exact mechanism is unclear, but may be due to the contributions of dystocia to abnormal placental separation. Proteolytic activity is hypothesized to contribute to placental separation in ruminants; however, when ovine placentomes were collected following caesarian section, no proteolytic activity was detected. We hypothesized that stage 2 of parturition was necessary to stimulate proteolysis and initiate placental separation. Serial placentome collections were performed on mature cows (n = 21 initiated; 7 with complete sampling) at hourly intervals for the first 2 h after expulsion of the calf. An intact piece of each placentome was fixed for histological evaluation, and a separate piece of caruncular and cotyledonary tissue from each placentome was frozen for transcriptomic and proteolytic analysis. A full set of placentomes was collected from only 7 of 21 cows at 0, 1, and 2 h, and all cows had expelled fetal membranes by 6 h. Histological, transcriptomic and proteolytic analysis was performed on placentomes from cows from which three placentomes were collected (n = 7). The microscopic distance between maternal and fetal tissues increased at 1 h (P = 0.01). Relative transcript abundance of matrix metalloprotease 14 (MMP14) tended to increase with time (P = 0.06). The relative transcript abundance of plasminogen activator urokinase-type (PLAU) was greater in caruncles than cotyledons (P = 0.01), and tended (P = 0.10) to increase in the caruncle between 0 and 2 h while remaining unchanged in the cotyledon over the same span of time. Greater PLAU and plasminogen activator tissue-type (PLAT) proteolytic activity was detected by zymography in the caruncle than the cotyledon immediately post-partum (P < 0.01). From these findings we conclude that 1) dystocia during the first parity decreases lifetime productivity in beef cattle, 2) the PA system is present at both the transcript and protein level in the bovine plactentome during parturition and 3) proteolytic activity is localized to the caruncular aspect of the placentome. Published by Elsevier Inc.

4G/5G Plasminogen Activator Inhibitor-1 Polymorphisms and Haplotypes Are Associated with Pneumonia

PubMed Central

Yende, Sachin; Angus, Derek C.; Ding, Jingzhong; Newman, Anne B.; Kellum, John A.; Li, Rongling; Ferrell, Robert E.; Zmuda, Joseph; Kritchevsky, Stephen B.; Harris, Tamara B.; Garcia, Melissa; Yaffe, Kristine; Wunderink, Richard G.

2007-01-01

Rationale: Plasminogen activator inhibitor (PAI)-1 inhibits urokinase and tissue plasminogen activator, required for host response to infection. Whether variation within the PAI-1 gene is associated with increased susceptibility to infection is unknown. Objectives: To ascertain the role of the 4G/5G polymorphism and other genetic variants within the PAI-1 gene. We hypothesized that variants associated with increased PAI-1 expression would be associated with an increased occurrence of community-acquired pneumonia (CAP). Methods: Longitudinal analysis (>12 yr) of the Health, Aging, and Body Composition cohort, aged 65–74 years at start of analysis. Measurements and Main Results: We genotyped the 4G/5G PAI-1 polymorphism and six additional single nucleotide polymorphisms. Of the 3,075 subjects, 272 (8.8%) had at least one hospitalization for CAP. Among whites, variants at the PAI4G,5G, PAI2846, and PAI7343 sites had higher risk of CAP (P = 0.018, 0.021, and 0.021, respectively). At these sites, variants associated with higher PAI-1 expression were associated with increased CAP susceptibility. Compared with the 5G/5G genotypes at PAI4G,5G site, the 4G/4G and 4G/5G genotypes were associated with a 1.98-fold increased risk of CAP (95% confidence interval, 1.2–3.2; P = 0.006). In whole blood stimulation assay, subjects with a 4G allele had 3.3- and 1.9-fold increased PAI-1 expression (P = 0.043 and 0.034, respectively). In haplotype analysis, the 4G/G/C/A haplotype at the PAI4G,5G, PAI2846, PAI4588, and PAI7343 single nucleotide polymorphisms was associated with higher CAP susceptibility, whereas the 5G/G/C/A haplotype was associated with lower CAP susceptibility. No associations were seen among blacks. Conclusions: Genotypes associated with increased expression of PAI-1 were associated with increased susceptibility to CAP in elderly whites. PMID:17761618

Safe and Effective Sarcoma Therapy through Bispecific Targeting of EGFR and uPAR

PubMed Central

Borgatti, Antonella; Koopmeiners, Joseph S.; Sarver, Aaron L.; Winter, Amber L.; Stuebner, Kathleen; Todhunter, Deborah; Rizzardi, Anthony E.; Henriksen, Jonathan C.; Schmechel, Stephen; Forster, Colleen L.; Kim, Jong-Hyuk; Froelich, Jerry; Walz, Jillian; Henson, Michael S.; Breen, Matthew; Lindblad-Toh, Kerstin; Oh, Felix; Pilbeam, Kristy; Modiano, Jaime F.; Vallera, Daniel A.

2017-01-01

Sarcomas differ from carcinomas in their mesenchymal origin. Therapeutic advancements have come slowly so alternative drugs and models are urgently needed. These studies report a new drug for sarcomas that simultaneously targets both tumor and tumor neovasculature. eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to epidermal growth factor (EGF) and the amino terminal fragment (ATF) of urokinase. Here, we study the drug in an in vivo “ontarget” companion dog trial since eBAT effectively kills canine hemangiosarcoma (HSA) and human sarcoma cells in vitro. We reasoned the model has value due to the common occurrence of spontaneous sarcomas in dogs and a limited lifespan allowing for rapid accrual and data collection. Splenectomized dogs with minimal residual disease were given one cycle of eBAT followed by adjuvant doxorubicin in an adaptive dose-finding, phase I–II study of 23 dogs with spontaneous, stage I–II, splenic HSA. eBAT improved 6-month survival from <40% in a comparison population to ~70% in dogs treated at a biologically active dose (50 μg/kg). Six dogs were long-term survivors, living >450 days. eBAT abated expected toxicity associated with EGFR-targeting, a finding supported by mouse studies. Urokinase plasminogen activator receptor (uPAR) and EGFR are targets for human sarcomas, so thorough evaluation is crucial for validation of the dog model. Thus, we validated these markers for human sarcoma targeting in the study of 212 human and 97 canine sarcoma samples. Our results support further translation of eBAT for human patients with sarcomas and perhaps other EGFR-expressing malignancies. PMID:28193671

The pharmacological modulation of thrombin-induced cerebral thromboembolism in the rabbit.

PubMed Central

May, G. R.; Paul, W.; Crook, P.; Butler, K. D.; Page, C. P.

1992-01-01

1. Intracarotid (i.c.) administration of thrombin induced a marked accumulation of 111indium-labelled platelets and 125I-labelled fibrinogen within the cranial vasculature of anaesthetized rabbits. 2. Thrombin (100 iu kg-1, i.c.) - induced platelet accumulation was completely abolished by pretreatment with desulphatohirudin (CGP 39393; 1 mg kg-1 i.c., 1 min prior to thrombin). Administration of CGP 39393 1 or 20 min after thrombin produced a significant reduction in platelet accumulation. 3. Intravenous (i.v.) administration of the platelet activating factor (PAF) receptor antagonist BN 52021 (10 mg kg-1) 5 min prior to thrombin (100 iu kg-1, i.c.) had no effect on platelet accumulation. 4. An inhibitor of NO biosynthesis, L-NG-nitro arginine methyl ester (L-NAME; 100 mg kg-1, i.c.), had no significant effect on the cranial platelet accumulation response to thrombin (10 iu kg-1, i.c.) when administered 5 min prior to thrombin. 5. Defibrotide (32 or 64 mg kg-1 bolus i.c. followed by 32 or 64 mg kg-1 h-1, i.c., infusion for 45 min) treatment begun 20 min after thrombin (100 iu kg-1, i.c.) did not significantly modify the cranial platelet accumulation response. 6. Cranial platelet accumulation induced by thrombin (100 iu kg-1, i.c.) was significantly reversed by the fibrinolytic drugs urokinase (20 iu kg-1, i.c., infusion for 45 min), anisoylated plasminogen streptokinase activator complex (APSAC) (200 micrograms kg-1, i.v. bolus) or recombinant tissue plasminogen activator (rt-PA; 100 micrograms kg-1, i.c. bolus followed by 20 micrograms kg-1 min-1, i.c., infusion for 45 min) administered 20 min after thrombin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1504722

Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages

PubMed Central

Graziano, Francesca; Desdouits, Marion; Garzetti, Livia; Podini, Paola; Alfano, Massimo; Rubartelli, Anna; Furlan, Roberto; Benaroch, Philippe; Poli, Guido

2015-01-01

HIV type 1 (HIV-1) infects CD4+ T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as “Trojan horses” carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages. PMID:26056317

Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages.

PubMed

Graziano, Francesca; Desdouits, Marion; Garzetti, Livia; Podini, Paola; Alfano, Massimo; Rubartelli, Anna; Furlan, Roberto; Benaroch, Philippe; Poli, Guido

2015-06-23

HIV type 1 (HIV-1) infects CD4(+) T lymphocytes and tissue macrophages. Infected macrophages differ from T cells in terms of decreased to absent cytopathicity and for active accumulation of new progeny HIV-1 virions in virus-containing compartments (VCC). For these reasons, infected macrophages are believed to act as "Trojan horses" carrying infectious particles to be released on cell necrosis or functional stimulation. Here we explored the hypothesis that extracellular ATP (eATP) could represent a microenvironmental signal potentially affecting virion release from VCC of infected macrophages. Indeed, eATP triggered the rapid release of infectious HIV-1 from primary human monocyte-derived macrophages (MDM) acutely infected with the CCR5-dependent HIV-1 strain. A similar phenomenon was observed in chronically infected promonocytic U1 cells differentiated to macrophage-like cells (D-U1) by costimulation with phorbol esters and urokinase-type plasminogen activator. Worthy of note, eATP did not cause necrotic, apoptotic, or pyroptotic cell death, and its effect on HIV-1 release was suppressed by Imipramine (an antidepressant agent known to inhibit microvesicle formation by interfering with membrane-associated acid sphingomyelinase). Virion release was not triggered by oxidized ATP, whereas the effect of eATP was inhibited by a specific inhibitor of the P2X7 receptor (P2X7R). Thus, eATP triggered the discharge of virions actively accumulating in VCC of infected macrophages via interaction with the P2X7R in the absence of significant cytopathicity. These findings suggest that the microvesicle pathway and P2X7R could represent exploitable targets for interfering with the VCC-associated reservoir of infectious HIV-1 virions in tissue macrophages.

PLAU inferred from a correlation network is critical for suppressor function of regulatory T cells

PubMed Central

He, Feng; Chen, Hairong; Probst-Kepper, Michael; Geffers, Robert; Eifes, Serge; del Sol, Antonio; Schughart, Klaus; Zeng, An-Ping; Balling, Rudi

2012-01-01

Human FOXP3+CD25+CD4+ regulatory T cells (Tregs) are essential to the maintenance of immune homeostasis. Several genes are known to be important for murine Tregs, but for human Tregs the genes and underlying molecular networks controlling the suppressor function still largely remain unclear. Here, we describe a strategy to identify the key genes directly from an undirected correlation network which we reconstruct from a very high time-resolution (HTR) transcriptome during the activation of human Tregs/CD4+ T-effector cells. We show that a predicted top-ranked new key gene PLAU (the plasminogen activator urokinase) is important for the suppressor function of both human and murine Tregs. Further analysis unveils that PLAU is particularly important for memory Tregs and that PLAU mediates Treg suppressor function via STAT5 and ERK signaling pathways. Our study demonstrates the potential for identifying novel key genes for complex dynamic biological processes using a network strategy based on HTR data, and reveals a critical role for PLAU in Treg suppressor function. PMID:23169000

Early breast cancer screening using iron/iron oxide-based nanoplatforms with sub-femtomolar limits of detection.

PubMed

Udukala, Dinusha N; Wang, Hongwang; Wendel, Sebastian O; Malalasekera, Aruni P; Samarakoon, Thilani N; Yapa, Asanka S; Abayaweera, Gayani; Basel, Matthew T; Maynez, Pamela; Ortega, Raquel; Toledo, Yubisela; Bossmann, Leonie; Robinson, Colette; Janik, Katharine E; Koper, Olga B; Li, Ping; Motamedi, Massoud; Higgins, Daniel A; Gadbury, Gary; Zhu, Gaohong; Troyer, Deryl L; Bossmann, Stefan H

2016-01-01

Proteases, including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins (CTS) exhibit numerous functions in tumor biology. Solid tumors are characterized by changes in protease expression levels by tumor and surrounding tissue. Therefore, monitoring protease levels in tissue samples and liquid biopsies is a vital strategy for early cancer detection. Water-dispersable Fe/Fe3O4-core/shell based nanoplatforms for protease detection are capable of detecting protease activity down to sub-femtomolar limits of detection. They feature one dye (tetrakis(carboxyphenyl)porphyrin (TCPP)) that is tethered to the central nanoparticle by means of a protease-cleavable consensus sequence and a second dye (Cy 5.5) that is directly linked. Based on the protease activities of urokinase plasminogen activator (uPA), MMPs 1, 2, 3, 7, 9, and 13, as well as CTS B and L, human breast cancer can be detected at stage I by means of a simple serum test. By monitoring CTS B and L stage 0 detection may be achieved. This initial study, comprised of 46 breast cancer patients and 20 apparently healthy human subjects, demonstrates the feasibility of protease-activity-based liquid biopsies for early cancer diagnosis.

Early breast cancer screening using iron/iron oxide-based nanoplatforms with sub-femtomolar limits of detection

PubMed Central

Samarakoon, Thilani N; Yapa, Asanka S; Abayaweera, Gayani; Basel, Matthew T; Maynez, Pamela; Ortega, Raquel; Toledo, Yubisela; Bossmann, Leonie; Robinson, Colette; Janik, Katharine E; Koper, Olga B; Li, Ping; Motamedi, Massoud; Higgins, Daniel A; Gadbury, Gary

2016-01-01

Summary Proteases, including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins (CTS) exhibit numerous functions in tumor biology. Solid tumors are characterized by changes in protease expression levels by tumor and surrounding tissue. Therefore, monitoring protease levels in tissue samples and liquid biopsies is a vital strategy for early cancer detection. Water-dispersable Fe/Fe3O4-core/shell based nanoplatforms for protease detection are capable of detecting protease activity down to sub-femtomolar limits of detection. They feature one dye (tetrakis(carboxyphenyl)porphyrin (TCPP)) that is tethered to the central nanoparticle by means of a protease-cleavable consensus sequence and a second dye (Cy 5.5) that is directly linked. Based on the protease activities of urokinase plasminogen activator (uPA), MMPs 1, 2, 3, 7, 9, and 13, as well as CTS B and L, human breast cancer can be detected at stage I by means of a simple serum test. By monitoring CTS B and L stage 0 detection may be achieved. This initial study, comprised of 46 breast cancer patients and 20 apparently healthy human subjects, demonstrates the feasibility of protease-activity-based liquid biopsies for early cancer diagnosis. PMID:27335730

Development of immunoassays for human urokinase

NASA Technical Reports Server (NTRS)

Atassi, M. Zouhair

1988-01-01

Radioimmune assays (RIA) and enzyme linked immune assays for measurement of pro-urokinase and the two active forms of the enzyme were developed. Polyclonal and monoclonal antibodies, with desired specificities against preselected synthetic regions of urokinase (UK), were obtained by immunization with the respective synthetic peptides and used to develop RIA for zymogen and the two activated forms of UK.

Phage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein.

PubMed

Urban, Johannes H; Moosmeier, Markus A; Aumüller, Tobias; Thein, Marcus; Bosma, Tjibbe; Rink, Rick; Groth, Katharina; Zulley, Moritz; Siegers, Katja; Tissot, Kathrin; Moll, Gert N; Prassler, Josef

2017-11-15

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products with drug-like properties. To fully exploit the potential of RiPPs as peptide drug candidates, tools for their systematic engineering are required. Here we report the engineering of lanthipeptides, a subclass of RiPPs characterized by multiple thioether cycles that are enzymatically introduced in a regio- and stereospecific manner, by phage display. This was achieved by heterologous co-expression of linear lanthipeptide precursors fused to the widely neglected C-terminus of the bacteriophage M13 minor coat protein pIII, rather than the conventionally used N-terminus, along with the modifying enzymes from distantly related bacteria. We observe that C-terminal precursor peptide fusions to pIII are enzymatically modified in the cytoplasm of the producing cell and subsequently displayed as mature cyclic peptides on the phage surface. Biopanning of large C-terminal display libraries readily identifies artificial lanthipeptide ligands specific to urokinase plasminogen activator (uPA) and streptavidin.

Impaired factor XIIa-dependent activation of fibrinolysis in treated antiphospholipid syndrome gestations developing late-pregnancy complications.

PubMed

Carmona, Francisco; Lázaro, Isabel; Reverter, Juan C; Tàssies, Dolors; Font, Josep; Cervera, Ricard; Balasch, Juan

2006-02-01

The objective of the study was to investigate the potential role of impaired factor XII-dependent activation of fibrinolysis in treated antiphospholipid syndrome gestations developing late-pregnancy complications. This was a prospective study in a third-level teaching hospital, including 75 patients: 25 pregnant patients having the antiphospholipid syndrome and carrying their pregnancies until 26 weeks' gestation or later (group 1); 25 pregnant patients having normal term pregnancies and delivery and no previous miscarriage (group 2); and 25 pregnant patients being diagnosed as having severe pre-eclampsia and/or intrauterine growth restriction but testing negative for antiphospholipid antibodies (group 3). Hemostatic evaluation was carried out from patients in groups 1 and 2 between 6 and 10 weeks, between 18 and 22 weeks, and between 28 and 32 weeks' gestation. Patients in group 3 were sampled between 28 and 32 weeks. An additional blood sample was obtained 4 to 6 months after delivery (baseline). The Mann-Whitney U test, the Friedman test, and the chi2 test were used. Patients in group 1 were characterized by increased factor VIIa levels, increased prothrombin fragment 1+2 levels, reduced factor XIIa levels, diminished functional urokinase-type plasminogen activator levels, and decreased levels of plasmin/alpha-2-plasmin inhibitor complexes. These abnormalities were more evident in patients in group 1 developing pre-eclampsia and/or intrauterine growth restriction. Impaired factor XIIa-dependent activation of fibrinolysis seems to be a key mechanism related to late-pregnancy complications in patients with the antiphospholipid syndrome.

The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

PubMed

Ehnman, M; Li, H; Fredriksson, L; Pietras, K; Eriksson, U

2009-01-29

Members of the platelet-derived growth factor (PDGF) family are mitogens for cells of mesenchymal origin and have important functions during embryonic development, blood vessel maturation, fibrotic diseases and cancer. In contrast to the two classical PDGFs, the novel and less well-characterized members, PDGF-CC and PDGF-DD, are latent factors that need to be processed extracellularly by activating proteases, before they can mediate PDGF receptor activation. Here, we elucidate the structural requirements for urokinase plasminogen activator (uPA)-mediated activation of PDGF-DD, as well as the intricate interplay with uPA receptor (uPAR) signalling. Furthermore, we show that activated PDGF-DD, in comparison to latent, more potently transforms NIH/3T3 cells in vitro. Conversely, xenograft studies in nude mice demonstrate that cells expressing latent PDGF-DD are more tumorigenic than those expressing activated PDGF-DD. These findings imply that a fine-tuned proteolytic activation, in the local milieu, controls PDGF-DD bioavailability. Moreover, we suggest that proteolytic activation of PDGF-DD reveals a retention motif mediating interactions with pericellular components. Our proposed mechanism, where uPA not only generates active PDGF-DD, but also regulates its spatial distribution, provides novel insights into the biological function of PDGF-DD.

Cell surface antigens in renal tumour cells: detection by immunoluminescence and enzymatic analysis

PubMed Central

Laube, F; Göhring, B; Sann, H; Willhardt, I

2001-01-01

Two renal cell carcinoma cell lines (49RC 43STR and 75RC 2STR) were characterized by detection of the cell surface proteins: CD44(var), intercellular adhesion molecule-1 (ICAM-1), urokinase-type plasminogen activator (uPA) and its receptor and aminopeptidase N (APN). To detect their localization the immunoluminescent technique was used. In addition, the enzyme activity of uPA and APN was investigated in cell suspensions as well as in monolayers. The latter procedure was more advantageous since the additional use of HPLC permits a single registration of the fluorescent hydrolysis-product AMC (7-amino-4-methylcoumarin) without interference by cellular autofluorescence or non-reacted fluorescent substrate. Unlike 75RC 2STR, the cell line 49RC 43STR expressed high levels of uPA and APN. Contrary to that the cell line 75RC 2STR expressed high levels of ICAM-1 and CD44(v6), whereas 49RC 43STR showed a low level of ICAM-1 and no distinct light signal with anti-CD44(v6). The uPA activity was measured directly as well as indirectly (via plasmin) with the substrate Z-Gly-Gly-Arg-AMC. Both activator and plasmin activity were inhibited by D-Val-Phe-Lys-CMK and phenylmethylsulfonyl fluoride. The anti-catalytic antibody to uPA and that to uPA receptor were found to be inhibiting the uPA activity in a concentration-dependent manner. APN activity was assayed using alanine-p-nitroanilide. Peptidase activity was effectively inhibited by 1,10-phenanthroline and partly inhibited by ethylenediamine-tetraacetic acid. © 2001 Cancer Research Campaignhttp://www.bjcancer.com PMID:11556847

A Cyclic Peptidic Serine Protease Inhibitor: Increasing Affinity by Increasing Peptide Flexibility

PubMed Central

Jiang, Longguang; Paaske, Berit; Kromann-Hansen, Tobias; Jensen, Jan K.; Sørensen, Hans Peter; Liu, Zhuo; Nielsen, Jakob T.; Christensen, Anni; Hosseini, Masood; Sørensen, Kasper K.; Nielsen, Niels Christian; Jensen, Knud J.; Huang, Mingdong; Andreasen, Peter A.

2014-01-01

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden. PMID:25545505

The effects of residual platelets in plasma on plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays.

PubMed

Pieters, Marlien; Barnard, Sunelle A; Loots, Du Toit; Rijken, Dingeman C

2017-01-01

Due to controversial evidence in the literature pertaining to the activity of plasminogen activator inhibitor-1 in platelets, we examined the effects of residual platelets present in plasma (a potential pre-analytical variable) on various plasminogen activator inhibitor-1 and plasminogen activator inhibitor-1-related assays. Blood samples were collected from 151 individuals and centrifuged at 352 and 1500 g to obtain plasma with varying numbers of platelet. In a follow-up study, blood samples were collected from an additional 23 individuals, from whom platelet-poor (2000 g), platelet-containing (352 g) and platelet-rich plasma (200 g) were prepared and analysed as fresh-frozen and after five defrost-refreeze cycles (to determine the contribution of in vitro platelet degradation). Plasminogen activator inhibitor-1 activity, plasminogen activator inhibitor-1 antigen, tissue plasminogen activator/plasminogen activator inhibitor-1 complex, plasma clot lysis time, β-thromboglobulin and plasma platelet count were analysed. Platelet α-granule release (plasma β-thromboglobulin) showed a significant association with plasminogen activator inhibitor-1 antigen levels but weak associations with plasminogen activator inhibitor-1 activity and a functional marker of fibrinolysis, clot lysis time. Upon dividing the study population into quartiles based on β-thromboglobulin levels, plasminogen activator inhibitor-1 antigen increased significantly across the quartiles while plasminogen activator inhibitor-1 activity and clot lysis time tended to increase in the 4th quartile only. In the follow-up study, plasma plasminogen activator inhibitor-1 antigen was also significantly influenced by platelet count in a concentration-dependent manner. Plasma plasminogen activator inhibitor-1 antigen levels increased further after complete platelet degradation. Residual platelets in plasma significantly influence plasma plasminogen activator inhibitor-1 antigen levels mainly through release of latent plasminogen activator inhibitor-1 with limited effects on plasminogen activator inhibitor-1 activity, tissue plasminogen activator/plasminogen activator inhibitor-1 complex or plasma clot lysis time. Platelets may however also have functional effects on plasma fibrinolytic potential in the presence of high platelet counts, such as in platelet-rich plasma.

Cadherin Switching and Activation of β-Catenin Signaling Underlie Proinvasive Actions of Calcitonin-Calcitonin Receptor Axis in Prostate Cancer*S⃞

PubMed Central

Shah, Girish V.; Muralidharan, Anbalagan; Gokulgandhi, Mitan; Soan, Kamal; Thomas, Shibu

2009-01-01

Calcitonin, a neuroendocrine peptide, and its receptor are localized in the basal epithelium of benign prostate but in the secretory epithelium of malignant prostates. The abundance of calcitonin and calcitonin receptor mRNA displays positive correlation with the Gleason grade of primary prostate cancers. Moreover, calcitonin increases tumorigenicity and invasiveness of multiple prostate cancer cell lines by cyclic AMP-dependent protein kinase-mediated actions. These actions include increased secretion of matrix metalloproteinases and urokinase-type plasminogen activator and an increase in prostate cancer cell invasion. Activation of calcitonin-calcitonin receptor autocrine loop in prostate cancer cell lines led to the loss of cell-cell adhesion, destabilization of tight and adherens junctions, and internalization of key integral membrane proteins. In addition, the activation of calcitonin-calcitonin receptor axis induced epithelial-mesenchymal transition of prostate cancer cells as characterized by cadherin switch and the expression of the mesenchymal marker, vimentin. The activated calcitonin receptor phosphorylated glycogen synthase kinase-3, a key regulator of cytosolic β-catenin degradation within the WNT signaling pathway. This resulted in the accumulation of intracellular β-catenin, its translocation in the nucleus, and transactivation of β-catenin-responsive genes. These results for the first time identify actions of calcitonin-calcitonin receptor axis on prostate cancer cells that lead to the destabilization of cell-cell junctions, epithelial-to-mesenchymal transition, and activation of WNT/β-catenin signaling. The results also suggest that cyclic AMP-dependent protein kinase plays a key role in calcitonin receptor-induced destabilization of cell-cell junctions and activation of WNT-β-catenin signaling. PMID:19001380

Molecular-specific urokinase antibodies

NASA Technical Reports Server (NTRS)

Atassi, M. Zouhair (Inventor); Morrison, Dennis R. (Inventor)

2009-01-01

Antibodies have been developed against the different molecular forms of urokinase using synthetic peptides as immunogens. The peptides were synthesized specifically to represent those regions of the urokinase molecules which are exposed in the three-dimensional configuration of the molecule and are uniquely homologous to urokinase. Antibodies are directed against the lysine 158-isoleucine 159 peptide bond which is cleaved during activation from the single-chain (ScuPA) form to the bioactive double chain (54 KDa and 33 KDa) forms of urokinase and against the lysine 135 lysine 136 bond that is cleaved in the process of removing the alpha-chain from the 54 KDa form to produce the 33 KDa form of urokinase. These antibodies enable the direct measurement of the different molecular forms of urokinase from small samples of conditioned medium harvested from cell cultures.

Inhibitory effects of Physalis angulata on tumor metastasis and angiogenesis.

PubMed

Hseu, You-Cheng; Wu, Chi-Rei; Chang, Hsueh-Wei; Kumar, K J Senthil; Lin, Ming-Kuem; Chen, Chih-Sheng; Cho, Hsin-Ju; Huang, Chun-Yin; Huang, Chih-Yang; Lee, Hong-Zin; Hsieh, Wen-Tsong; Chung, Jing-Gung; Wang, Hui-Min; Yang, Hsin-Ling

2011-06-01

ETHNOPHARMACOLOGICAL RELAVENCE: Physalis angulata is well-known in traditional Chinese medicine as a ingredient for various herbal formulation; also, it has been shown to exhibit anti-cancer and anti-inflammatory effects. In this study, the ability of P. angulata to inhibit tumor metastasis and angiogenesis was investigated. Anti-proliferative activity of ethyl acetate extracts of P. angulata (PA extracts), was determined against human oral squamous carcinoma (HSC-3) and human umbilical vein endothelial cells (HUVECs) by trypan blue exclusion method. Wound-healing migration, trans-well invasion, Western blotting and chick chorioallantoic membrane assay were carried out to determine the anti-metastatic and anti-angiogenic effects of PA extracts in vitro and in vivo. We demonstrated that at sub-cytotoxic concentrations of PA extracts (5-15 μg/mL) markedly inhibited the migration and invasion of highly metastatic HSC-3 cells as shown by wound-healing repair assay and trans-well assay. Gelatin zymography assay showed that PA extracts suppressed the activity of matrix metalloproteinase (MMP)-9 and -2, and urokinase plasminogen activator (u-PA) in HSC-3 cells. In addition, Western blot analysis confirmed that PA extracts significantly decreased MMP-2 and u-PA protein expression in HSC-3 cells. Notably, PA extracts significantly augmented the expression of their endogenous inhibitors, including tissue inhibitors of MMP (TIMP-1 and -2), and plasminogen activator inhibitors (PAI-1 and -2). Further investigations revealed that non-cytotoxic concentration of PA extracts (5-15 μg/mL) inhibited vascular endothelial growth factor (VEGF)-induced proliferation, and migration/invasion of HUVECs in vitro. PA extracts also suppressed the activity of MMP-9, but not MMP-2, in HUVECs. Further, we observed, PA extracts strongly suppressed neovessel formation in the chorioallantoic membrane of chick embryos in vivo. These results strongly support an anti-metastatic and anti-angiogenic activity of P. angulata that may contribute to the development of better chemopreventive agent for cancer and inflammation. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

High-level expression of a novel recombinant human plasminogen activator (rhPA) in the milk of transgenic rabbits and its thrombolytic bioactivity in vitro.

PubMed

Song, Shaozheng; Ge, Xin; Cheng, Yaobin; Lu, Rui; Zhang, Ting; Yu, Baoli; Ji, Xueqiao; Qi, Zhengqiang; Rong, Yao; Yuan, Yuguo; Cheng, Yong

2016-08-01

The human tissue-type plasminogen activator (tPA) is a key kinase of fibrinolysis that plays an important role in dissolving fibrin clots to promote thrombolysis. The recombinant human plasminogen activator (rhPA) has more thrombolytic advantages than the wild type tPA. To increase the half-life and thrombolytic activity of tPA, a mutant containing only the essential K2 fibrin-binding and P activating plasminogen domains of the wild type tPA was cloned. This fragment was then inserted into goat β-casein regulatory sequences. Then, a mammary gland-specific expression vector, PCL25/rhPA, was constructed, and the transgenic rabbits were generated. In this study, 18 live transgenic founders (12♀, 6♂) were generated using pronuclear microinjection. Six transgenic rabbits were obtained, and the expression levels of rhPA in the milk had a range of 15.2-630 µg/ml. A fibrin agarose plate assay of rhPA showed that it had strong thrombolytic bioactivity in vitro, and the highest specific activity was >360 (360 times more than that of alteplase). The results indicated that the rhPA containing only the K2 and P domains is efficiently expressed with higher thrombolytic bioactivity in the milk of transgenic rabbits. Our study also demonstrated a new method for the large-scale production of clinically relevant recombinant pharmaceutical proteins in the mammary glands of transgenic rabbits.

Myelin basic protein stimulates plasminogen activation via tissue plasminogen activator following binding to independent l-lysine-containing domains.

PubMed

Gonzalez-Gronow, Mario; Fiedler, Jenny L; Farias Gomez, Cristian; Wang, Fang; Ray, Rupa; Ferrell, Paul D; Pizzo, Salvatore V

2017-08-26

Myelin basic protein (MBP) is a key component of myelin, the specialized lipid membrane that encases the axons of all neurons. Both plasminogen (Pg) and tissue-type plasminogen activator (t-PA) bind to MBP with high affinity. We investigated the kinetics and mechanisms involved in this process using immobilized MBP and found that Pg activation by t-PA is significantly stimulated by MBP. This mechanism involves the binding of t-PA via a lysine-dependent mechanism to the Lys 91 residue of the MBP NH 2 -terminal region Asp 82 -Pro 99 , and the binding of Pg via a lysine-dependent mechanism to the Lys 122 residue of the MBP COOH-terminal region Leu 109 -Gly 126 . In this context, MBP mimics fibrin and because MBP is a plasmin substrate, our results suggest direct participation of the Pg activation system on MBP physiology. Copyright © 2017 Elsevier Inc. All rights reserved.

Ginger extract diminishes chronic fructose consumption-induced kidney injury through suppression of renal overexpression of proinflammatory cytokines in rats.

PubMed

Yang, Ming; Liu, Changjin; Jiang, Jian; Zuo, Guowei; Lin, Xuemei; Yamahara, Johji; Wang, Jianwei; Li, Yuhao

2014-05-27

The metabolic syndrome is associated with an increased risk of development and progression of chronic kidney disease. Renal inflammation is well known to play an important role in the initiation and progression of tubulointerstitial injury of the kidneys. Ginger, one of the most commonly used spices and medicinal plants, has been demonstrated to improve diet-induced metabolic abnormalities. However, the efficacy of ginger on the metabolic syndrome-associated kidney injury remains unknown. This study aimed to investigate the impact of ginger on fructose consumption-induced adverse effects in the kidneys. The fructose control rats were treated with 10% fructose in drinking water over 5 weeks. The fructose consumption in ginger-treated rats was adjusted to match that of fructose control group. The ethanolic extract of ginger was co-administered (once daily by oral gavage). The indexes of lipid and glucose homeostasis were determined enzymatically, by ELISA and/or histologically. Gene expression was analyzed by Real-Time PCR. In addition to improve hyperinsulinemia and hypertriglyceridemia, supplement with ginger extract (50 mg/kg) attenuated liquid fructose-induced kidney injury as characterized by focal cast formation, slough and dilation of tubular epithelial cells in the cortex of the kidneys in rats. Furthermore, ginger also diminished excessive renal interstitial collagen deposit. By Real-Time PCR, renal gene expression profiles revealed that ginger suppressed fructose-stimulated monocyte chemoattractant protein-1 and its receptor chemokine (C-C motif) receptor-2. In accord, overexpression of two important macrophage accumulation markers CD68 and F4/80 was downregulated. Moreover, overexpressed tumor necrosis factor-alpha, interleukin-6, transforming growth factor-beta1 and plasminogen activator inhibitor (PAI)-1 were downregulated. Ginger treatment also restored the downregulated ratio of urokinase-type plasminogen activator to PAI-1. The present results suggest that ginger supplement diminishes fructose-induced kidney injury through suppression of renal overexpression of macrophage-associated proinflammatory cytokines in rats. Our findings provide evidence supporting the protective effect of ginger on the metabolic syndrome-associated kidney injury.

Global Gene Expression Profiling in PAI-1 Knockout Murine Heart and Kidney: Molecular Basis of Cardiac-Selective Fibrosis

PubMed Central

Ghosh, Asish K.; Murphy, Sheila B.; Kishore, Raj; Vaughan, Douglas E.

2013-01-01

Fibrosis is defined as an abnormal matrix remodeling due to excessive synthesis and accumulation of extracellular matrix proteins in tissues during wound healing or in response to chemical, mechanical and immunological stresses. At present, there is no effective therapy for organ fibrosis. Previous studies demonstrated that aged plasminogen activator inhibitor-1(PAI-1) knockout mice develop spontaneously cardiac-selective fibrosis without affecting any other organs. We hypothesized that differential expressions of profibrotic and antifibrotic genes in PAI-1 knockout hearts and unaffected organs lead to cardiac selective fibrosis. In order to address this prediction, we have used a genome-wide gene expression profiling of transcripts derived from aged PAI-1 knockout hearts and kidneys. The variations of global gene expression profiling were compared within four groups: wildtype heart vs. knockout heart; wildtype kidney vs. knockout kidney; knockout heart vs. knockout kidney and wildtype heart vs. wildtype kidney. Analysis of illumina-based microarray data revealed that several genes involved in different biological processes such as immune system processing, response to stress, cytokine signaling, cell proliferation, adhesion, migration, matrix organization and transcriptional regulation were affected in hearts and kidneys by the absence of PAI-1, a potent inhibitor of urokinase and tissue-type plasminogen activator. Importantly, the expressions of a number of genes, involved in profibrotic pathways including Ankrd1, Pi16, Egr1, Scx, Timp1, Timp2, Klf6, Loxl1 and Klotho, were deregulated in PAI-1 knockout hearts compared to wildtype hearts and PAI-1 knockout kidneys. While the levels of Ankrd1, Pi16 and Timp1 proteins were elevated during EndMT, the level of Timp4 protein was decreased. To our knowledge, this is the first comprehensive report on the influence of PAI-1 on global gene expression profiling in the heart and kidney and its implication in fibrogenesis and several other biological processes. The significance of these observations in the light of heart-specific profibrotic signaling and fibrogenesis are discussed. PMID:23724005

Ginger extract diminishes chronic fructose consumption-induced kidney injury through suppression of renal overexpression of proinflammatory cytokines in rats

PubMed Central

2014-01-01

Background The metabolic syndrome is associated with an increased risk of development and progression of chronic kidney disease. Renal inflammation is well known to play an important role in the initiation and progression of tubulointerstitial injury of the kidneys. Ginger, one of the most commonly used spices and medicinal plants, has been demonstrated to improve diet-induced metabolic abnormalities. However, the efficacy of ginger on the metabolic syndrome-associated kidney injury remains unknown. This study aimed to investigate the impact of ginger on fructose consumption-induced adverse effects in the kidneys. Methods The fructose control rats were treated with 10% fructose in drinking water over 5 weeks. The fructose consumption in ginger-treated rats was adjusted to match that of fructose control group. The ethanolic extract of ginger was co-administered (once daily by oral gavage). The indexes of lipid and glucose homeostasis were determined enzymatically, by ELISA and/or histologically. Gene expression was analyzed by Real-Time PCR. Results In addition to improve hyperinsulinemia and hypertriglyceridemia, supplement with ginger extract (50 mg/kg) attenuated liquid fructose-induced kidney injury as characterized by focal cast formation, slough and dilation of tubular epithelial cells in the cortex of the kidneys in rats. Furthermore, ginger also diminished excessive renal interstitial collagen deposit. By Real-Time PCR, renal gene expression profiles revealed that ginger suppressed fructose-stimulated monocyte chemoattractant protein-1 and its receptor chemokine (C-C motif) receptor-2. In accord, overexpression of two important macrophage accumulation markers CD68 and F4/80 was downregulated. Moreover, overexpressed tumor necrosis factor-alpha, interleukin-6, transforming growth factor-beta1 and plasminogen activator inhibitor (PAI)-1 were downregulated. Ginger treatment also restored the downregulated ratio of urokinase-type plasminogen activator to PAI-1. Conclusions The present results suggest that ginger supplement diminishes fructose-induced kidney injury through suppression of renal overexpression of macrophage-associated proinflammatory cytokines in rats. Our findings provide evidence supporting the protective effect of ginger on the metabolic syndrome-associated kidney injury. PMID:24885946

MMPs-Mediated ECM Remodeling

PubMed

Mali, Aniket V; Joshi, Asavari A; Hegde, Mahabaleshwar V; Kadam, Shivajirao S

2017-04-01

Background: To enhance their own survival, tumor cells can manipulate their microenvironment through remodeling of the extra cellular matrix (ECM). The urokinase-type plasminogen activator (uPA) system catalyzes plasmin production which further mediates activation of matrix metalloproteinases (MMPs) and plays an important role in breast cancer invasion and metastasis through ECM remodeling. This provides a potential target for therapeutic intervention of breast cancer treatment. Enterolactone (EL) is derived from dietary flax lignans in the human body and is known to have anti-breast cancer activity. We here investigated molecular and cellular mechanisms of EL action on the uPA-plasmin- MMPs system. Methods: MTT and trypan blue dye exclusion assays, anchorage-dependent clonogenic assays and wound healing assays were carried out to study effects on cell proliferation and viability, clonogenicity and migration capacity, respectively. Real-time PCR was employed to study gene expression and gelatin zymography was used to assess MMP-2 and MMP-9 activities. All data were statistically analysed and presented as mean ± SEM values. Results: All the findings collectively demonstrated anticancer and antimetastatic potential of EL with antiproliferative, antimigratory and anticlonogenic cellular mechanisms. EL was found to exhibit multiple control of plasmin activation by down-regulating uPA expression and also up-regulating its natural inhibitor, PAI-1, at the mRNA level. Further, EL was found to down-regulate expression of MMP-2 and MMP-9 genes, and up-regulate TIMP-1 and TIMP-2; natural inhibitors of MMP-2 and MMP-9, respectively. This may be as a consequence of inhibition of plasmin activation, resulting in robust control over migration and invasion of breast cancer cells during metastasis. Conclusions: EL suppresses proliferation, migration and metastasis of MDA-MB-231 breast cancer cells by inhibiting induced ECM remodeling by the ‘uPA-plasmin-MMPs system’. Creative Commons Attribution License

Intra-tubular hydrodynamic forces influence tubulo-interstitial fibrosis in the kidney

PubMed Central

Rohatgi, Rajeev; Flores, Daniel

2010-01-01

Purpose of review Renal epithelial cells respond to mechanical stimuli with immediate transduction events (e.g., activation of ion channels), intermediate biological responses (e.g., changes in gene expression), and long term cellular adaptation (e.g., protein expression). Progressive renal disease is characterized by disturbed glomerular hydrodynamics that contributes to glomerulosclerosis, but, how intra-tubular biomechanical forces contribute to tubulo-interstital inflammation and fibrosis is poorly understood. Recent findings In vivo and in vitro models of obstructive uropathy demonstrate that tubular stretch induces robust expression of transforming growth factor β-1 (TGFβ-1), activation of tubular apoptosis, and induction of NF-κB signaling which contribute to the inflammatory and fibrotic milieu. Non-obstructive structural kidney diseases associated with nephron loss follow a course characterized by compensatory increases of single nephron glomerular filtration rate and tubular flow rate. Resulting increases in tubular fluid shear stress (FSS) reduce tissue-plasminogen activator and urokinase enzymatic activity which diminishes breakdown of extracellular matrix. In models of high dietary Na intake, which increase tubular flow, urinary TGFβ-1 concentrations and renal mitogen activated protein kinase activity are increased. Summary In conclusion, intra-tubular biomechanical forces, stretch and FSS, generate changes in intracellular signaling and gene expression that contribute to the pathobiology of obstructive, and non-obstructive kidney disease. PMID:19851105

Intravenous thrombolysis guided by a telemedicine consultation system for acute ischaemic stroke patients in China: the protocol of a multicentre historically controlled study

PubMed Central

Yuan, Ziwen; Wang, Bo; Li, Feijiang; Wang, Jing; Zhi, Jin; Luo, Erping; Liu, Zhirong; Zhao, Gang

2015-01-01

Introduction The rate of intravenous thrombolysis with tissue-type plasminogen activator or urokinase for stroke patients is extremely low in China. It has been demonstrated that a telestroke service may help to increase the rate of intravenous thrombolysis and improve stroke care quality in local hospitals. The aim of this study, also called the Acute Stroke Advancing Program, is to evaluate the effectiveness and safety of decision-making concerning intravenous thrombolysis via a telemedicine consultation system for acute ischaemic stroke patients in China. Methods and analysis This is a multicentre historically controlled study with a planned enrolment of 300 participants in each of two groups. The telestroke network consists of one hub hospital and 14 spoke hospitals in underserved regions of China. The usual stroke care quality in the spoke hospitals without guidance from the hub hospital will be used as the historical control. The telemedicine consultation system is an interactive, two-way, wireless, audiovisual system accessed on portable devices. The primary outcome is the percentage of patients treated with intravenous thrombolysis within 4.5 h of stroke onset. Ethics and dissemination The project has been approved by the Institutional Review Board of Xijing Hospital. The results will be published in scientific journals and presented to local government and relevant institutes. Trial registration number NCT02088346 (12 March 2014). PMID:25979867

β-Catenin—A Linchpin in Colorectal Carcinogenesis?

PubMed Central

Wong, Newton Alexander Chiang Shuek; Pignatelli, Massimo

2002-01-01

An important role for β-catenin pathways in colorectal carcinogenesis was first suggested by the protein’s association with adenomatous polyposis coli (APC) protein, and by evidence of dysregulation of β-catenin protein expression at all stages of the adenoma-carcinoma sequence. Recent studies have, however, shown that yet more components of colorectal carcinogenesis are linked to β-catenin pathways. Pro-oncogenic factors that also release β-catenin from the adherens complex and/or encourage translocation to the nucleus include ras, epidermal growth factor (EGF), c-erbB-2, PKC-βΙΙ, MUC1, and PPAR-γ, whereas anti-oncogenic factors that also inhibit nuclear β-catenin signaling include transforming growth factor (TGF)-β, retinoic acid, and vitamin D. Association of nuclear β-catenin with the T cell factor (TCF)/lymphoid enhancer factor (LEF) family of transcription factors promotes the expression of several compounds that have important roles in the development and progression of colorectal carcinoma, namely: c-myc, cyclin D1, gastrin, cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-7, urokinase-type plasminogen activator receptor (aPAR), CD44 proteins, and P-glycoprotein. Finally, genetic aberrations of several components of the β-catenin pathways, eg, Frizzled (Frz), AXIN, and TCF-4, may potentially contribute to colorectal carcinogenesis. In discussing the above interactions, this review demonstrates that β-catenin represents a key molecule in the development of colorectal carcinoma. PMID:11839557

Use of quantitative optical imaging to examine the role of cholesterol-rich lipid raft microdomains in the migration of breast cancer cells

NASA Astrophysics Data System (ADS)

You, Minghai; Chen, Jianling; Wang, Shaobing; Dong, Shiqing; Wang, Yuhua; Xie, Shusen; Wang, Zhengchao; Yang, Hongqin

2018-04-01

Lipid rafts have been extensively studied and shown to be involved in many cancers, including breast cancer. However, the exact role of lipid rafts in the migration of breast cancer cells remains unclear. This study was designed to examine lipid rafts (cholesterol) in the plasma membrane of breast cancer cells (MDA-MB-231 and MCF-7) and normal breast epithelial cells (MCF-10A) through generalized polarization values, and further investigate the role of cholesterol-rich lipid rafts in the migration of breast cancer cells. The results showed that the plasma membrane in breast cancer cells was more orderly than that in normal epithelial cells; this was correlated with expression changes of matrix metallopeptidase 9 (MMP-9) and urokinase-type plasminogen activator receptor (uPAR), the markers of cancer cell migration. Moreover, the breast cancer cells were more sensitive to the reagent that induced cholesterol depletion than the normal breast epithelial cells, while the capacity of cancer cells to migrate decreased significantly according to changes in MMP-9 and uPAR expression. To our best knowledge, this is the first demonstration of the relationship between cholesterol-rich lipid rafts and the migration of breast cancer cells; it could be useful for the prevention of breast cancer and early treatment through reduction of the level of cholesterol in the plasma membrane of the cells.

PAI-1 promoter 4G/5G polymorphism (rs1799768) contributes to tumor susceptibility: Evidence from meta-analysis.

PubMed

Xu, Xin; Xie, Yanqi; Lin, Yiwei; Xu, Xianglai; Zhu, Yi; Mao, Yeqing; Hu, Zhenghui; Wu, Jian; Chen, Hong; Zheng, Xiangyi; Qin, Jie; Xie, Liping

2012-12-01

Plasminogen activator inhibitor-1 (PAI-1), belonging to the urokinase plasminogen activation (uPA) system, is involved in cancer development and progression. The PAI-1 promoter 4G/5G polymorphism was shown to contribute to genetic susceptibility to cancer, although the results were inconsistent. To assess this relationship more precisely, a meta-analysis was performed. The electronic databases PubMed, Scopus, Web of Science and Chinese National Knowledge Infrastructure (CNKI) were searched; data were extracted and analyzed independently by two reviewers. Ultimately, 21 eligible case-control studies with a total of 8,415 cancer cases and 9,208 controls were included. The overall odds ratio (OR) with its 95% confidence interval (CI) showed a statistically significant association between the PAI-1 promoter 4G/5G polymorphism and cancer risk (4G/4G vs. 5G/5G: OR=1.25, 95% CI=1.07-1.47, P(heterogeneity)=0.001; 4G/4G vs. 4G/5G+5G/5G: OR=1.10, 95% CI=1.03-1.17, P(heterogeneity)=0.194; 4G/4G+4G/5G vs. 5G/5G: OR=1.17, 95% CI=1.01-1.35, P(heterogeneity)=0.041). In further subgroup analyses, the increased risk of cancer was observed in a subgroup of Caucasians with regards to endometrial cancer. Our meta-analysis suggests that the PAI-1 4G/5G polymorphism most likely contributes to susceptibility to cancer, particularly in Caucasians. Furthermore, the 4G allele may be associated with an increased risk of endometrial cancer.

PAI-1 promoter 4G/5G polymorphism (rs1799768) contributes to tumor susceptibility: Evidence from meta-analysis

PubMed Central

XU, XIN; XIE, YANQI; LIN, YIWEI; XU, XIANGLAI; ZHU, YI; MAO, YEQING; HU, ZHENGHUI; WU, JIAN; CHEN, HONG; ZHENG, XIANGYI; QIN, JIE; XIE, LIPING

2012-01-01

Plasminogen activator inhibitor-1 (PAI-1), belonging to the urokinase plasminogen activation (uPA) system, is involved in cancer development and progression. The PAI-1 promoter 4G/5G polymorphism was shown to contribute to genetic susceptibility to cancer, although the results were inconsistent. To assess this relationship more precisely, a meta-analysis was performed. The electronic databases PubMed, Scopus, Web of Science and Chinese National Knowledge Infrastructure (CNKI) were searched; data were extracted and analyzed independently by two reviewers. Ultimately, 21 eligible case-control studies with a total of 8,415 cancer cases and 9,208 controls were included. The overall odds ratio (OR) with its 95% confidence interval (CI) showed a statistically significant association between the PAI-1 promoter 4G/5G polymorphism and cancer risk (4G/4G vs. 5G/5G: OR=1.25, 95% CI=1.07–1.47, Pheterogeneity=0.001; 4G/4G vs. 4G/5G+5G/5G: OR=1.10, 95% CI=1.03–1.17, Pheterogeneity=0.194; 4G/4G+4G/5G vs. 5G/5G: OR=1.17, 95% CI=1.01–1.35, Pheterogeneity=0.041). In further subgroup analyses, the increased risk of cancer was observed in a subgroup of Caucasians with regards to endometrial cancer. Our meta-analysis suggests that the PAI-1 4G/5G polymorphism most likely contributes to susceptibility to cancer, particularly in Caucasians. Furthermore, the 4G allele may be associated with an increased risk of endometrial cancer. PMID:23226787

Effects of a high-fat diet on spontaneous metastasis of Lewis lung carcinoma in plasminogen activator inhibitor-1 deficient and wild-type mice

USDA-ARS?s Scientific Manuscript database

We investigated the effects of plasminogen activator inhibitor-1 (PAI-1) deficiency on spontaneous metastasis of Lewis lung carcinoma (LLC) in PAI-1 deficient (PAI-1-/-) and wildtype mice (C57BL/6J background) fed the AIN93G diet or that diet modified with 45% calories from fat. The high-fat diet i...

Gingival crevicular fluid tissue/blood vessel-type plasminogen activator and plasminogen activator inhibitor-2 levels in patients with rheumatoid arthritis: effects of nonsurgical periodontal therapy.

PubMed

Kurgan, Ş; Önder, C; Balcı, N; Fentoğlu, Ö; Eser, F; Balseven, M; Serdar, M A; Tatakis, D N; Günhan, M

2017-06-01

The aim of this study was to evaluate the effect of nonsurgical periodontal therapy on clinical parameters and gingival crevicular fluid levels of tissue/blood vessel-type plasminogen activator (t-PA) and plasminogen activator inhibitor-2 (PAI-2) in patients with periodontitis, with or without rheumatoid arthritis (RA). Fifteen patients with RA and chronic periodontitis (RA-P), 15 systemically healthy patients with chronic periodontitis (H-P) and 15 periodontally and systemically healthy volunteers (C) were included in the study. Plaque index, gingival index, probing pocket depth, clinical attachment level, bleeding on probing, gingival crevicular fluid t-PA and PAI-2 levels, erythrocyte sedimentation rate, serum C-reactive protein and disease activity score were evaluated at baseline and 3 mo after mechanical nonsurgical periodontal therapy. All periodontal clinical parameters were significantly higher in the RA-P and H-P groups compared with the C group (p < 0.001) and decreased significantly after treatment (p < 0.001). Pretreatment t-PA levels were highest in the RA-P group and significantly decreased post-treatment (p = 0.047). Pre- and post-treatment PAI-2 levels were significantly lower in controls compared with both periodontitis groups (p < 0.05). Gingival crevicular fluid volume and the levels of t-PA and PAI-2 were significantly correlated. In patients with periodontitis and RA, nonsurgical periodontal therapy reduced the pretreatment gingival crevicular fluid t-PA levels, which were significantly correlated with gingival crevicular fluid PAI-2 levels. The significantly higher t-PA and PAI-2 gingival crevicular fluid levels in periodontal patients, regardless of systemic status, suggest that the plasminogen activating system plays a role in the disease process of periodontitis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Dose-ranging study of the novel recombinant plasminogen activator BM 06.022 in healthy volunteers.

PubMed

Martin, U; von Möllendorff, E; Akpan, W; Kientsch-Engel, R; Kaufmann, B; Neugebauer, G

1991-10-01

The novel recombinant plasminogen activator BM 06.022 consists of the kringle 2 and protease domains of human tissue-type plasminogen activator and is unglycosylated because of its expression in Escherichia coli cells. Pharmacokinetics for activity and hemostatic effects of BM 06.022 were studied in 18 healthy male volunteers after an intravenous bolus injection over 2 minutes. BM 06.022 was administered successively at doses of 0.1125, 0.55, 2.2, 3.3, 4.4, and 5.5 MU to three volunteers. Plasma fibrinogen was unchanged; effects of BM 06.022 were observed on plasminogen only at higher doses, and dose-dependent effects were seen on alpha 2-antiplasmin and fibrin D-dimers. The concentration of plasminogen and alpha 2-antiplasmin was 87% +/- 3% and 79% +/- 3%, respectively, of baseline 2 hours after injection of 5.5 MU of BM 06.022. Fibrin D-dimers were highest with 1147 +/- 380 ng/ml at 5.5 MU of BM 06.022. The area under the activity concentration-time curve (AUC) increased dose-dependently and linearly. At 5.5 MU of BM 06.022, the AUC was 313 +/- 47 IU.hr.ml-1, the total plasma clearance was 306 +/- 40 ml/min, and the half-life was 14.4 +/- 1.1 minutes.

12/15-Lipoxygenase Inhibition or Knockout Reduces Warfarin-Associated Hemorrhagic Transformation After Experimental Stroke.

PubMed

Liu, Yu; Zheng, Yi; Karatas, Hulya; Wang, Xiaoying; Foerch, Christian; Lo, Eng H; van Leyen, Klaus

2017-02-01

For stroke prevention, patients with atrial fibrillation typically receive oral anticoagulation. The commonly used anticoagulant warfarin increases the risk of hemorrhagic transformation (HT) when a stroke occurs; tissue-type plasminogen activator treatment is therefore restricted in these patients. This study was designed to test the hypothesis that 12/15-lipoxygenase (12/15-LOX) inhibition would reduce HT in warfarin-treated mice subjected to experimental stroke. Warfarin was dosed orally in drinking water, and international normalized ratio values were determined using a Coaguchek device. C57BL6J mice or 12/15-LOX knockout mice were subjected to transient middle cerebral artery occlusion with 3 hours severe ischemia (model A) or 2 hours ischemia and tissue-type plasminogen activator infusion (model B), with or without the 12/15-LOX inhibitor ML351. Hemoglobin was determined in brain homogenates, and hemorrhage areas on the brain surface and in brain sections were measured. 12/15-LOX expression was detected by immunohistochemistry. Warfarin treatment resulted in reproducible increased international normalized ratio values and significant HT in both models. 12/15-LOX knockout mice suffered less HT after severe ischemia, and ML351 reduced HT in wild-type mice. When normalized to infarct size, ML351 still independently reduced hemorrhage. HT after tissue-type plasminogen activator was similarly reduced by ML351. In addition to its benefits in infarct size reduction, 12/15-LOX inhibition also may independently reduce HT in warfarin-treated mice. ML351 should be further evaluated as stroke treatment in anticoagulated patients suffering a stroke, either alone or in conjunction with tissue-type plasminogen activator. © 2017 American Heart Association, Inc.

The interplay between tissue plasminogen activator domains and fibrin structures in the regulation of fibrinolysis: kinetic and microscopic studies

PubMed Central

Thelwell, Craig; Williams, Stella C.; Silva, Marta M. C. G.; Szabó, László; Kolev, Krasimir

2011-01-01

Regulation of tissue-type plasminogen activator (tPA) depends on fibrin binding and fibrin structure. tPA structure/function relationships were investigated in fibrin formed by high or low thrombin concentrations to produce a fine mesh and small pores, or thick fibers and coarse structure, respectively. Kinetics studies were performed to investigate plasminogen activation and fibrinolysis in the 2 types of fibrin, using wild-type tPA (F-G-K1-K2-P, F and K2 binding), K1K1-tPA (F-G-K1-K1-P, F binding), and delF-tPA (G-K1-K2-P, K2 binding). There was a trend of enzyme potency of tPA > K1K1-tPA > delF-tPA, highlighting the importance of the finger domain in regulating activity, but the differences were less apparent in fine fibrin. Fine fibrin was a better surface for plasminogen activation but more resistant to lysis. Scanning electron and confocal microscopy using orange fluorescent fibrin with green fluorescent protein-labeled tPA variants showed that tPA was strongly associated with agglomerates in coarse but not in fine fibrin. In later lytic stages, delF-tPA-green fluorescent protein diffused more rapidly through fibrin in contrast to full-length tPA, highlighting the importance of finger domain-agglomerate interactions. Thus, the regulation of fibrinolysis depends on the starting nature of fibrin fibers and complex dynamic interaction between tPA and fibrin structures that vary over time. PMID:20966169

A nutrient mixture inhibits glioblastoma xenograft U-87 MG growth in male nude mice.

PubMed

Roomi, M W; Kalinovsky, T; Rath, M; Niedzwiecki, A

2016-03-01

Brain tumors are highly aggressive tumors characterized by secretions of high levels of matrix metalloproteinase-2 and -9, leading to tumor growth, invasion and metastasis by digesting the basement membrane and extracellular matrix components. We previously demonstrated the effectiveness of a nutrient mixture (NM) containing ascorbic acid, lysine, proline, and green tea extract in vitro: on activity of urokinase plasminogen activator, matrix metalloproteinases and TIMPs in various human glioblastoma (LN-18, T-98G and A-172) cell lines and on glioblastoma A-172 cell proliferation and Matrigel invasion. Our main objective in this study was to investigate the effect of the NM in vivo on human glioblastoma U-87 MG cell line. Athymic male nude mice inoculated with 3·10(6) U-87 MG cells subcutaneously and were fed a regular diet or a regular diet supplemented with 0.5% NM. Four weeks later, the mice were sacrificed, the tumors were weighed and measured. The samples were studied histologically. NM inhibited tumor weight and tumor burden by 53% (p = 0.015) and 48% (p = 0.010), respectively. These results suggest the therapeutic potential of NM as an adjuvant in the treatment of glioblastoma.

[Disseminated intravascular coagulation induced by endotoxin in rabbits: effect of treatment with t-PA and urokinase].

PubMed

Paloma, M J; Páramo, J A; Rifón, J; Rocha, E

1992-12-01

To assess the therapeutic efficacy of agents capable of stimulating the fibrinolytic system, such as tissue plasminogen activator (t-PA) and urokinase (UK) on endotoxin-induced disseminated intravascular coagulation (DIC) in the rabbit. DIC was induced by intravenous administration of endotoxin, 20 micrograms/kg/hr during 6 hr. Four different groups were established: a) control group, receiving only saline solution; b) t-PA group receiving 0.2 mg/kg; c) t-PA group receiving 0.7 mg/kg, and d) UK group, which was given 3,000 IU/kg/hr for 6 hr. Blood samples were drawn before and after 2 hr and 6 hr of endotoxin administration. Platelet count, and fibrinogen, factor XII and antithrombin III concentrations, were assessed in each sample. Mean, standard deviation and percentage of increase or decrease with respect to the basal value, this considered 100%, were used to evaluate the findings. For comparison of values, Student's t and Mann Whitney's U were used; the Fisher test was used for mortality studies. No statistical differences appeared for any of the values in the rabbits under basal conditions. The rabbits in the control group developed DIC. No doses of t-PA modified the changes appearing in blood coagulation. UK reduced the fibrinogen and factor XII consumption induced by endotoxin. The mortality rate in the control group reached 70%. High-dose t-PA decreased such figure to 50%, while low-dose t-PA or UK failed to reduce mortality. High-dose t-PA has beneficial effects on endotoxin-induced DIC in rabbits. UK failed to achieve such effect at the doses given in this experimental DIC model.

CYP1A1 and CYP1A2 expression: Comparing ‘humanized’ mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

PubMed Central

Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

2009-01-01

Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how “human-like” can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1_CYP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+)_severe-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs. PMID:19285097

CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji

2009-05-15

Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1{sub C}YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carryingmore » humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+){sub s}evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.« less

Twelve-Month Clinical and Quality-of-Life Outcomes in the Interventional Management of Stroke III Trial.

PubMed

Palesch, Yuko Y; Yeatts, Sharon D; Tomsick, Thomas A; Foster, Lydia D; Demchuk, Andrew M; Khatri, Pooja; Hill, Michael D; Jauch, Edward C; Jovin, Tudor G; Yan, Bernard; von Kummer, Rüdiger; Molina, Carlos A; Goyal, Mayank; Schonewille, Wouter J; Mazighi, Mikael; Engelter, Stefan T; Anderson, Craig; Spilker, Judith; Carrozzella, Janice; Ryckborst, Karla J; Janis, L Scott; Simpson, Annie; Simpson, Kit N; Broderick, Joseph P

2015-05-01

Randomized trials have indicated a benefit for endovascular therapy in appropriately selected stroke patients at 3 months, but data regarding outcomes at 12 months are currently lacking. We compared functional and quality-of-life outcomes at 12 months overall and by stroke severity in stroke patients treated with intravenous tissue-type plasminogen activator followed by endovascular treatment as compared with intravenous tissue-type plasminogen activator alone in the Interventional Management of Stroke III Trial. The key outcome measures were a modified Rankin Scale score ≤2 (functional independence) and the Euro-QoL EQ-5D, a health-related quality-of-life measure. 656 subjects with moderate-to-severe stroke (National Institutes of Health Stroke Scale ≥8) were enrolled at 58 centers in the United States (41 sites), Canada (7), Australia (4), and Europe (6). There was an interaction between treatment group and stroke severity in the repeated measures analysis of modified Rankin Scale ≤2 outcome (P=0.039). In the 204 participants with severe stroke (National Institutes of Health Stroke Scale ≥20), a greater proportion of the endovascular group had a modified Rankin Scale ≤2 (32.5%) at 12 months as compared with the intravenous tissue-type plasminogen activator group (18.6%, P=0.037); no difference was seen for the 452 participants with moderately severe strokes (55.6% versus 57.7%). In participants with severe stroke, the endovascular group had 35.2 (95% confidence interval: 2.1, 73.3) more quality-adjusted-days over 12 months as compared with intravenous tissue-type plasminogen activator alone. Endovascular therapy improves functional outcome and health-related quality-of-life at 12 months after severe ischemic stroke. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00359424. © 2015 American Heart Association, Inc.

Optical heterogeneous bioassay for the detection of the inflammatory biomarker suPAR

NASA Astrophysics Data System (ADS)

Tombelli, S.; Trono, C.; Adinolfi, B.; Chiavaioli, F.; Giannetti, A.; Eugen-Olsen, J.; Bernini, R.; Grimaldi, I. A.; Persichetti, G.; Testa, G.; Baldini, F.

2015-03-01

Soluble urokinase plasminogen activator receptor (suPAR) is an inflammatory protein present in blood and a marker of disease presence, severity and prognosis. A heterogeneous sandwich assay is proposed for quantifying suPAR by employing a capture antibody from rat and a biotinylated detection antibody from mouse. Optical detection was achieved by a successive exposure of the biotinylated sandwich to streptavidin labelled with ATTO647N. The heterogeneous assay was implemented on a multichannel polymethylmetacrylate (PMMA) optical biochip, potentially capable of the simultaneous detection of more than one analyte. Capture antibody was immobilized on the PMMA surface of the microfluidic channel and the assay was performed with the following protocol: i) surface blocking with BSA, ii) incubation with suPAR or PBS, iii) incubation with biotinylated suPAR detection Ab and iv) incubation with streptavidin-ATTO647N. Promising preliminary results were obtained with this protocol. Moreover, an improved optical setup is proposed which avoids the mechanical scanning of the chip and consequently the in-series fluorescence excitation and read out, allowing the simultaneous measurement of the fluorescence on all the channels of the microfluidic chip.

Burn Injury Alters Epidermal Cholinergic Mediators and Increases HMGB1 and Caspase 3 in Autologous Donor Skin and Burn Margin

PubMed Central

Holmes, Casey J.; Plichta, Jennifer K.; Gamelli, Richard L.; Radek, Katherine A.

2016-01-01

Burn wound healing complications, such as graft failure or infection, are a major source of morbidity and mortality in burn patients. The mechanisms by which local burn injury alters epidermal barrier function in autologous donor skin and surrounding burn margin are largely undefined. We hypothesized that defects in the epidermal cholinergic system may impair epidermal barrier function and innate immune responses. The objective was to identify alterations in the epidermal cholinergic pathway, and their downstream targets, associated with inflammation and cell death. We established that protein levels, but not gene expression, of the α7 nicotinic acetylcholine receptor (CHRNA7) were significantly reduced in both donor and burn margin skin. Furthermore, the gene and protein levels of an endogenous allosteric modulator of CHRNA7, secreted mammalian Ly-6/urokinase-type plasminogen activator receptor-related protein-1 (SLURP1) and acetylcholine were significantly elevated in donor and burn margin skin. As downstream proteins of inflammatory and cell death targets of nAChR activation, we found significant elevations in epidermal High Mobility Group Box Protein 1 (HMGB1) and caspase 3 in donor and burn margin skin. Lastly, we employed a novel in vitro keratinocyte burn model to establish that burn injury influences the gene expression of these cholinergic mediators and their downstream targets. These results indicate that defects in cholinergic mediators and inflammatory/apoptotic molecules in donor and burn margin skin may directly contribute to graft failure or infection in burn patients. PMID:27648692

The activation of plasminogen by Hageman factor (Factor XII) and Hageman factor fragments.

PubMed Central

Goldsmith, G H; Saito, H; Ratnoff, O S

1978-01-01

Activation of plasminogen through surface-mediated reactions is well recognized. In the presence of kaolin, purified Hageman factor (Factor XII) changed plasminogen to plasmin, as assayed upon a synthetic amide substrate and by fibrinolysis. Kinetic studies suggested an enzymatic action of Hageman factor upon its substrate, plasminogen. Hageman factor fragments, at a protein concentration equivalent to whole Hageman factor, activated plasminogen to a lesser extent. These protein preparations were not contaminated with other agents implicated in surface-mediated fibrinolysis. Diisopropyl fluorophosphate treatment of plasminogen did not inhibit its activation by Hageman factor. These studies indicate that Hageman factor has a hitherto unsuspected function, the direct activation of plasminogen. PMID:659637

Curcumin Attenuates Staurosporine-Mediated Death of Retinal Ganglion Cells

PubMed Central

Burugula, Balabharathi; Ganesh, Bhagyalaxmi S.

2011-01-01

Purpose. Staurosporine (SS) causes retinal ganglion cell (RGC) death in vivo, but the underlying mechanisms have been unclear. Since previous studies on RGC-5 cells indicated that SS induces cell death by elevating proteases, this study was undertaken to investigate whether SS induces RGC loss by elevating proteases in the retina, and curcumin prevents SS-mediated death of RGCs. Methods. Transformed mouse retinal ganglion-like cells (RGC-5) were treated with 2.0 μM SS and various doses of curcumin. Two optimal doses of SS (12.5 and 100 nM) and curcumin (2.5 and 10 μM) were injected into the vitreous of C57BL/6 mice. Matrix metalloproteinase (MMP)-9, tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) activities were assessed by zymography assays. Viability of RGC-5 cells was assessed by MTT assays. RGC and amacrine cell loss in vivo was assessed by immunostaining with Brn3a and ChAT antibodies, respectively. Frozen retinal cross sections were immunostained for nuclear factor-κB (NF-κB). Results. Staurosporine induced uPA and tPA levels in RGC-5 cells, and MMP-9, uPA, and tPA levels in the retinas and promoted the death of RGC-5 cells in vitro and RGCs and amacrine cells in vivo. In contrast, curcumin attenuated RGC and amacrine cell loss, despite elevated levels of proteases. An NF-κB inhibitory peptide reversed curcumin-mediated protective effect on RGC-5 cells, but did not inhibit protease levels. Curcumin did not inhibit protease levels in vivo, but attenuated RGC and amacrine cell loss by restoring NF-κB expression. Conclusions. The results show that curcumin attenuates RGC and amacrine cell death despite elevated levels of proteases and raises the possibility that it may be used as a plausible adjuvant therapeutic agent to prevent the loss of these cells in retinal degenerative conditions. PMID:21498608

Thrombolytic effect of nattokinase on a chemically induced thrombosis model in rat.

PubMed

Fujita, M; Hong, K; Ito, Y; Fujii, R; Kariya, K; Nishimuro, S

1995-10-01

Nattokinase is a new fibrinolytic enzyme which cleaves directly cross-linked fibrin in vitro. In this study, we investigated the thrombolytic effect of nattokinase on a thrombus in the common carotid artery of rat in which the endothelial cells of the vessel wall were injured by acetic acid. When a section of occluded vessel was stained for CD61 antigen by immunofluorescence utilizing a monoclonal antibody, the antigen was localized around the surface of the occluded blood vessels. This result suggests that the occlusive thrombosis was caused by platelet aggregation. In addition, thrombolysis with urokinase (UK; 50000 IU/kg, i.v.) or tissue plasminogen activator (tPA; 13300 IU/kg, i.v.) in our model was observed to restore the blood flow over a 60 min monitoring period. The results indicate that our chemically induced model is useful for screening and evaluating a thrombolytic agent. We evaluated the thrombolytic activity of nattokinase using this model and compared it with fibrino(geno)lytic enzyme, plasmin or elastase. On a molar basis, the recovery of the arterial blood flow with nattokinase, plasmin and elastase were 62.0 +/- 5.3%, 15.8 +/- 0.7% and 0%, respectively. The results indicate that the thrombolytic activity of nattokinase is stronger than that of plasmin or elastase in vivo.

3-Hydroxyflavone inhibits human osteosarcoma U2OS and 143B cells metastasis by affecting EMT and repressing u-PA/MMP-2 via FAK-Src to MEK/ERK and RhoA/MLC2 pathways and reduces 143B tumor growth in vivo.

PubMed

Lu, Ko-Hsiu; Chen, Pei-Ni; Hsieh, Yi-Hsien; Lin, Chin-Yin; Cheng, Fu-Yuan; Chiu, Peng-Chou; Chu, Shu-Chen; Hsieh, Yih-Shou

2016-11-01

Many natural flavonoids have cytostatic and apoptotic properties; however, we little know whether the effect of synthetic 3-hydroxyflavone on metastasis and tumor growth of human osteosarcoma. Here, we tested the hypothesis that 3-hydroxyflavone suppresses human osteosarcoma cells metastasis and tumor growth. 3-hydroxyflavone, up to 50 μM without cytotoxicity, inhibited U2OS and 143B cells motility, invasiveness and migration by reducing matrix metalloproteinase (MMP)-2 and urokinase-type plasminogen activator (u-PA) and also impaired cell adhesion to gelatin. 3-hydroxyflavone significantly reduced p-focal adhesion kinase (FAK) Tyr397, p-FAK Tyr925, p-steroid receptor coactivator (Src), p-mitogen/extracellular signal-regulated kinase (MEK)1/2, p-myosin light chain (MLC)2 Ser19, epithelial cell adhesion molecule, Ras homolog gene family (Rho)A and fibronectin expressions. 3-hydroxyflavone also affected the epithelial-mesenchymal transition (EMT) by down-regulating expressions of Vimentin and α-catenin with activation of the transcription factor Slug. In nude mice xenograft model and tail vein injection model showed that 3-hydroxyflavone reduced 143B tumor growth and lung metastasis. 3-hydroxyflavone possesses the anti-metastatic activity of U2OS and 143B cells by affecting EMT and repressing u-PA/MMP-2 via FAK-Src to MEK/ERK and RhoA/MLC2 pathways and suppresses 143B tumor growth in vivo. This may lead to clinical trials of osteosarcoma chemotherapy to confirm the promising result in the future. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bombesin stimulates invasion and migration of Isreco1 colon carcinoma cells in a Rho-dependent manner.

PubMed

Saurin, Jean-Christophe; Fallavier, Marjorie; Sordat, Bernard; Gevrey, Jean-Claude; Chayvialle, Jean-Alain; Abello, Jacques

2002-08-15

The membrane receptor for the neuropeptide bombesin/gastrin-releasing peptide (GRP) is expressed by a large fraction of human colorectal carcinoma cells. We reported previously a stimulation of cell adhesion and lamellipodia formation by the neuropeptide bombesin in the human, bombesin/GRP receptor-expressing, Isreco1 colorectal cancer cell line (J. C. Saurin et al., Cancer Res., 59: 962-967, 1999). Using invasion and motility assays, we demonstrate in this report that bombesin can both enhance the invasive capacity of Isreco1 cells in a dose-dependent manner (maximal effect at 1 nM) and stimulate the closure of wounds performed on confluent Isreco1 cells. These effects were reversed fully by the specific bombesin/GRP receptor antagonist D-Phe(6)-Bn(6-13)OMe used at 1 micro M. MMP-9 and urokinase-type plasminogen activator were expressed by Isreco1 cells, and bombesin did not significantly alter their level of secretion. Interestingly, exoenzyme C3 (10 micro g/ml) decreased cell invasiveness induced by bombesin by 70% and completely inhibited the migration of Isreco1 cells. Similarly, the Rho-kinase inhibitor Y-27632 dose-dependently reduced the effect of bombesin on cell invasion. Moreover, pull-down assays for GTP-bound RhoA demonstrated that bombesin was able to activate the small G-protein in Isreco1 cells. These results show that the neuropeptide bombesin is able to modulate invasiveness of Isreco1 colorectal carcinoma cells in vitro through a Rho-dependent pathway, leading to an increase in cell locomotion without a significant effect on tumor-cell associated proteolytic activity. These findings indicate that bombesin/GRP receptor expression may contribute to the cellular events that are critical for invasion/migration of colorectal carcinoma cells.

An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eren, Elif; Murphy, Megan; Goguen, Jon

2010-08-13

The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changesmore » of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.« less

Regulation of Plasminogen Activation on Cell Surfaces and Fibrin.

PubMed

Urano, Tetsumei; Castellino, Francis J; Suzuki, Yuko

2018-05-20

The fibrinolytic system dissolves fibrin and maintains vascular patency. Recent advances in imaging analyses allowed visualization of the spatiotemporal regulatory mechanism of fibrinolysis, as well as its regulation by other plasma haemostasis cofactors. Vascular endothelial cells (VECs) retain tissue-type plasminogen activator (tPA) after secretion and maintain high plasminogen (plg) activation potential on their surfaces. As in plasma, the serpin, plasminogen activator inhibitor type 1 (PAI-1), regulates fibrinolytic potential via inhibition of the VEC surface-bound plg activator, tPA. Once fibrin is formed, plg activation by tPA is initiated and effectively amplified on the surface of fibrin, and fibrin is rapidly degraded. The specific binding of plg and tPA to lytic edges of partly degraded fibrin via newly generated C-terminal lysine residues, which amplifies fibrin digestion, is a central aspect of this pathophysiological mechanism. Thrombomodulin (TM) plays a role in the attenuation of the plg binding on fibrin and the associated fibrinolysis, which is reversed by a carboxypeptidase B inhibitor. This suggests that the plasma procarboxypeptidase B, thrombin activatable fibrinolysis inhibitor (TAFI), which is activated by thrombin bound to TM on VECs, is a critical aspect of the regulation of plg activation on VECs and subsequent fibrinolysis. Platelets also contain PAI-1, TAFI, TM and the fibrin crosslinking enzyme, Factor (F) XIIIa, and either secrete or expose these agents upon activation in order to regulate fibrinolysis. In this review, the native machinery of plg activation and fibrinolysis, as well as their spatiotemporal regulatory mechanisms, as revealed by imaging analyses, are discussed. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Production of Experimental Malignant Pleural Effusions Is Dependent on Invasion of the Pleura and Expression of Vascular Endothelial Growth Factor/Vascular Permeability Factor by Human Lung Cancer Cells

PubMed Central

Yano, Seiji; Shinohara, Hisashi; Herbst, Roy S.; Kuniyasu, Hiroki; Bucana, Corazon D.; Ellis, Lee M.; Fidler, Isaiah J.

2000-01-01

We determined the molecular mechanisms that regulate the pathogenesis of malignant pleural effusion (PE) associated with advanced stage of human, non-small-cell lung cancer. Intravenous injection of human PC14 and PC14PE6 (adenocarcinoma) or H226 (squamous cell carcinoma) cells into nude mice yielded numerous lung lesions. PC14 and PC14PE6 lung lesions invaded the pleura and produced PE containing a high level of vascular endothelial growth factor (VEGF)-localized vascular hyperpermeability. Lung lesions produced by H226 cells were confined to the lung parenchyma with no PE. The level of expression of VEGF mRNA and protein by the cell lines directly correlated with extent of PE formation. Transfection of PC14PE6 cells with antisense VEGF165 gene did not inhibit invasion into the pleural space but reduced PE formation. H226 cells transfected with either sense VEGF 165 or sense VEGF 121 genes induced localized vascular hyperpermeability and produced PE only after direct implantation into the thoracic cavity. The production of PE was thus associated with the ability of tumor cells to invade the pleura, a property associated with expression of high levels of urokinase-type plasminogen activator and low levels of TIMP-2. Collectively, the data demonstrate that the production of malignant PE requires tumor cells to invade the pleura and express high levels of VEGF/VPF. PMID:11106562

Pivotal role of tissue plasminogen activator in the mechanism of action of electroconvulsive therapy.

PubMed

Hoirisch-Clapauch, Silvia; Mezzasalma, Marco A U; Nardi, Antonio E

2014-02-01

Electroconvulsive therapy is an important treatment option for major depressive disorders, acute mania, mood disorders with psychotic features, and catatonia. Several hypotheses have been proposed as electroconvulsive therapy's mechanism of action. Our hypothesis involves many converging pathways facilitated by increased synthesis and release of tissue-plasminogen activator. Human and animal experiments have shown that tissue-plasminogen activator participates in many mechanisms of action of electroconvulsive therapy or its animal variant, electroconvulsive stimulus, including improved N-methyl-D-aspartate receptor-mediated signaling, activation of both brain-derived neurotrophic factor and vascular endothelial growth factor, increased bioavailability of zinc, purinergic release, and increased mobility of dendritic spines. As a result, tissue-plasminogen activator helps promote neurogenesis in limbic structures, modulates synaptic transmission and plasticity, improves cognitive function, and mediates antidepressant effects. Notably, electroconvulsive therapy seems to influence tissue-plasminogen activator metabolism. For example, electroconvulsive stimulus increases the expression of glutamate decarboxylase 65 isoform in γ-aminobutyric acid-releasing neurons, which enhances the release of tissue-plasminogen activator, and the expression of p11, a protein involved in plasminogen and tissue-plasminogen activator assembling. This paper reviews how electroconvulsive therapy correlates with tissue-plasminogen activator. We suggest that interventions aiming at increasing tissue-plasminogen activator levels or its bioavailability - such as daily aerobic exercises together with a carbohydrate-restricted diet, or normalization of homocysteine levels - be evaluated in controlled studies assessing response and remission duration in patients who undergo electroconvulsive therapy.

Thrombolysis based on magnetically-controlled surface-functionalized Fe3O4 nanoparticle

PubMed Central

Chang, Ming; Lin, Yu-Hao; Gabayno, Jacque Lynn; Li, Qian; Liu, Xiaojun

2017-01-01

ABSTRACT In this study, the control of magnetic fields to manipulate surface-functionalized Fe3O4 nanoparticles by urokinase coating is investigated for thrombolysis in a microfluidic channel. The urokinase-coated Fe3O4 nanoparticles are characterized using particle size distribution, zeta potential measurement and spectroscopic data. Thrombolytic ratio tests reveal that the efficiency for thrombus cleaning is significantly improved when using magnetically-controlled urokinase-coated Fe3O4 nanoparticles than pure urokinase solution. The average increase in the rate of thrombolysis with the use of urokinase-coated Fe3O4 nanoparticles is about 50%. In vitro thrombolysis test in a microfluidic channel using the coated nanoparticles shows nearly complete removal of thrombus, a result that can be attributed to the clot busting effect of the urokinase as it inhibits the possible formation of blood bolus during the magnetically-activated microablation process. The experiment further demonstrates that a thrombus mass of 10.32 mg in the microchannel is fully removed in about 180 s. PMID:27689864

History of drugs for thrombotic disease. Discovery, development, and directions for the future.

PubMed

Mueller, R L; Scheidt, S

1994-01-01

The history of the antithrombotic agents--aspirin, heparin, warfarin, and the thrombolytics--is a rich and lively odyssey of serendipity, perseverance, vision, and conflict involving a number of striking personalities. The history of aspirin spans ages and continents from Hippocrates' analgesic for women in labor to the rediscovery of the white willow bark by English country scholar Reverend Edward Stone. Bayer chemist Felix Hoffmann reinvented aspirin for his ailing father; suburban physician L.L. Craven pioneered the prophylactic antithrombotic uses of aspirin; and Sir John Vane elucidated aspirin's mechanism of action as the inhibition of prostaglandin synthetase. Heparin was discovered by McLean, working as a medical student in 1915 in search of a pure procoagulant in dog liver. His original impure material differed somewhat from today's heparin, but purified heparin was rapidly accepted for a myriad of clinical uses; to this day, diverse new properties of this complex glycosaminoglycan continue to be elucidated. The oral anticoagulants emerged from veterinary research in the 1920s on a hemorrhagic disorder afflicting cattle that consumed spoiled sweet clover hay. Several chance encounters led Karl Link and his University of Wisconsin team to the identification of dicumarol as the offending agent in 1939 and its widespread therapeutic use by Wright and others in the 1940s. Link later developed warfarin as a rodenticide, but its use in humans soon followed in the 1950s. Vitamin K was discovered in the 1930s; its involvement in the mechanism of the anticoagulant agents was not delineated until the 1970s. The intrinsic ability of clotted blood to liquify and the fibrinolytic properties of normal urine were noted in the 1800s. Tillett and Sherry's group stumbled on the fibrinolytic properties of streptokinase in the 1930s and pioneered the therapeutic use of streptokinase in the 1940s and of urokinase in the 1960s. Several teams found tissue-type plasminogen activator in various body sites beginning in the 1940s, leading to its cloning and widespread use in the 1980s; anisoylated plasminogen-streptokinase activator complex is an example of rational drug design. The discoverers of these diverse agents have not only provided physicians with a potent armamentarium of antithrombotic drugs but also helped elucidate much basic science and vividly demonstrated the merits of perseverance, independent thought, and adherance to the scientific method.

Enterolactone Suppresses Proliferation, Migration and Metastasis of MDA-MB-231 Breast Cancer Cells Through Inhibition of uPA Induced Plasmin Activation and MMPs-Mediated ECM Remodeling

PubMed Central

Mali, Aniket V; Joshi, Asavari A; Hegde, Mahabaleshwar V; Kadam, Shivajirao S

2017-01-01

Background: To enhance their own survival, tumor cells can manipulate their microenvironment through remodeling of the extra cellular matrix (ECM). The urokinase-type plasminogen activator (uPA) system catalyzes plasmin production which further mediates activation of matrix metalloproteinases (MMPs) and plays an important role in breast cancer invasion and metastasis through ECM remodeling. This provides a potential target for therapeutic intervention of breast cancer treatment. Enterolactone (EL) is derived from dietary flax lignans in the human body and is known to have anti-breast cancer activity. We here investigated molecular and cellular mechanisms of EL action on the uPA-plasmin-MMPs system. Methods: MTT and trypan blue dye exclusion assays, anchorage-dependent clonogenic assays and wound healing assays were carried out to study effects on cell proliferation and viability, clonogenicity and migration capacity, respectively. Real-time PCR was employed to study gene expression and gelatin zymography was used to assess MMP-2 and MMP-9 activities. All data were statistically analysed and presented as mean ± SEM values. Results: All the findings collectively demonstrated anticancer and antimetastatic potential of EL with antiproliferative, antimigratory and anticlonogenic cellular mechanisms. EL was found to exhibit multiple control of plasmin activation by down-regulating uPA expression and also up-regulating its natural inhibitor, PAI-1, at the mRNA level. Further, EL was found to down-regulate expression of MMP-2 and MMP-9 genes, and up-regulate TIMP-1 and TIMP-2; natural inhibitors of MMP-2 and MMP-9, respectively. This may be as a consequence of inhibition of plasmin activation, resulting in robust control over migration and invasion of breast cancer cells during metastasis. Conclusions: EL suppresses proliferation, migration and metastasis of MDA-MB-231 breast cancer cells by inhibiting induced ECM remodeling by the ‘uPA-plasmin-MMPs system’. PMID:28545187

Effect of pH and glucose on cultured human peritoneal mesothelial cells.

PubMed

Shao, J C; Yorioka, N; Nishida, Y; Yamakido, M

1999-08-01

We investigated the effects of various pH and glucose concentrations on the growth of human peritoneal mesothelial cells and on coagulation and fibrinolytic factors. Cells were cultured at various pH values in Ham's F-12 medium containing 1.0% foetal calf serum and supplemented with D-glucose or D-mannitol at various concentrations. After 4-48 h, cell proliferation and 3H-thymidine incorporation were determined. Coagulation and fibrinolytic factors were measured after 48 h. Glucose caused concentration-dependent inhibition of cell growth at all pH values, but the deleterious effect of low pH on cell proliferation was faster and stronger than that of high glucose. At a similar osmolality, mannitol caused less inhibition of cell proliferation than glucose. There was a glucose concentration-dependent increase of thrombin-antithrombin III complex production at all pH values. At pH 5.2, tissue-type plasminogen activator production was far lower than at higher pH values, and production of the plasminogen activator inhibitor showed a glucose concentration-dependent increase. At pH 6.5 or 7.3, however, the plasminogen activator inhibitor production decreased and tissue-type plasminogen activator production increased in a glucose concentration-dependent manner. Low pH and/or high glucose culture medium had an inhibitory effect on peritoneal mesothelial cells, with the effect of high glucose being partially related to hyperosmolality. These cells may modulate peritoneal coagulant and fibrinolytic activity, with the balance between coagulation and fibrinolysis being disturbed by low pH and/or high glucose.

The pathophysiology of pelvic floor disorders: evidence from a histomorphologic study of the perineum and a mouse model of rectal prolapse

PubMed Central

YIOU, RENÉ; DELMAS, VINCENT; CARMELIET, PETER; GHERARDI, ROMAIN K.; BARLOVATZ-MEIMON, GEORGIA; CHOPIN, DOMINIQUE K.; ABBOU, CLÉMENT-CLAUDE; LEFAUCHEUR, JEAN-PASCAL

2001-01-01

The muscle changes related to pelvic floor disorders are poorly understood. We conducted an anatomical and histological study of the perineum of the normal mouse and of a transgenic mouse strain deficient in urokinase-type plasminogen activator (uPA−/−) that was previously reported to develop a high incidence of rectal prolapse. We could clearly identify the iliococcygeus (ILC) and pubococcygeus (PC) muscles and anal (SPA) and urethral (SPU) sphincters in male and female mice. The bulbocavernosus (BC), ischiocavernosus (ISC) and levator ani (LA) muscles could be found only in male mice. Histochemical analysis of the pelvic floor muscles revealed a majority of type IIA fibres. Rectal prolapses were observed only in male uPA−/− mice. The most obvious finding was an irreducible evagination of the rectal mucosa and a swelling of the entire perineal region corresponding to an irreducible hernia of the seminal vesicles through the pelvic outlet. The hernia caused stretching and thinning of the ISC, BC and LA. Myopathic damage, with degenerated and centronucleated myofibres, were observed in these muscles. The PC, ILC, SPA and SPU were not affected. This study provides an original description of a model of pelvic floor disorder and illustrates the differences existing between the perineum of humans and that of a quadruped species. In spite of these differences, the histopathologic changes observed in the pelvic floor muscles of uPA−/− mice with rectal prolapse suggest that prolonged muscular stretching causes a primary myopathic injury. This should be taken into account in the evaluation of pelvic floor disorders. PMID:11760891

Propagation of Human Hepatocytes in uPA/SCID Mice: Producing Chimeric Mice with Humanized Liver.

PubMed

Ohshita, Hiroki; Tateno, Chise

2017-01-01

Primary or cryopreserved human hepatocytes (h-heps) have been used as the gold standard for in vitro metabolism and hepatotoxicity studies; however, the supply of h-heps is limited and they cannot grow in vitro. We achieved approximately 1000-fold propagation of h-heps in the liver of albumin promoter/enhancer-driven urokinase-type plasminogen activator transgenic/severe combined immunodeficiency disease (uPA/SCID) mice with genetically induced liver disease and immunodeficiency. When h-heps are transplanted into the uPA/SCID mouse liver via the spleen, the h-heps engraft in the mouse liver, resulting in its repopulation with h-heps. We have named this model "chimeric mouse with humanized liver, PXB-mouse ® ." Fresh h-heps can be isolated from the chimeric mice (PXB-cells ® ) and have been used for in vitro studies.The efficacy and safety of chemical entities for use in humans are estimated using experimental animals such as rats and mice. The drug development of many chemical entities has been halted because of metabolic differences between humans and animals during clinical studies. Therefore, chimeric mice with humanized liver have been used to predict human-type metabolism and safety conditions for h-heps. In addition, until recently there were no suitable hepatitis B or C virus (HBV or HCV) susceptible animal models aside from chimpanzees. Chimeric mice are the sole persistent infectious small animal model for HBV and HCV and they have been used to investigate the efficacy of new anti-HBV or HCV agents.In this chapter, we describe a method for producing chimeric mice with humanized liver using uPA/SCID mice.

Epsilon-aminocaproic acid prevents high glucose and insulin induced-invasiveness in MDA-MB-231 breast cancer cells, modulating the plasminogen activator system.

PubMed

Viedma-Rodríguez, Rubí; Martínez-Hernández, María Guadalupe; Flores-López, Luis Antonio; Baiza-Gutman, Luis Arturo

2018-01-01

Obesity and type II diabetes mellitus have contributed to the increase of breast cancer incidence worldwide. High glucose concentration promotes the proliferation of metastatic cells, favoring the activation of the plasminogen/plasmin system, thus contributing to tumor progression. The efficient formation of plasmin is dependent on the binding of plasminogen to the cell surface. We studied the effect of ε-aminocaproic acid (EACA), an inhibitor of the binding of plasminogen to cell surface, on proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and plasminogen activation system, in metastatic MDA-MB-231 breast cancer cells grown in a high glucose microenvironment and treated with insulin. MDA-MB-231 cells were treated with EACA 12.5 mmol/L under high glucose 30 mmol/L (HG) and high glucose and insulin 80 nmol/L (HG-I) conditions, evaluating: cell population growth, % of viability, migratory, and invasive abilities, as well as the expression of uPA, its receptor (uPAR), and its inhibitor (PAI-1), by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, MMP-2 and MMP-9 mRNAs were evaluated by RT-PCR. Markers of EMT were evaluated by Western blot. Additionally, the presence of active uPA was studied by gel zymography, using casein-plasminogen as substrates. EACA prevented the increase in cell population, migration and invasion induced by HG and insulin, which was associated with the inhibition of EMT and the attenuation of HG- and insulin-dependent expression of uPA, uPAR, PAI-1, MMP-2, MMP-9, α-enolase (ENO A), and HCAM. The interaction of plasminogen to the cell surface and plasmin formation are mediators of the prometastasic action of hyperglycemia and insulin, potentially, EACA can be employed in the prevention and as adjuvant treatment of breast tumorigenesis promoted by hyperglycemia and insulin.

Computational analysis of blood clot dissolution using a vibrating catheter tip.

PubMed

Lee, Jeong Hyun; Oh, Jin Sun; Yoon, Bye Ri; Choi, Seung Hong; Rhee, Kyehan; Jho, Jae Young; Han, Moon Hee

2012-04-01

We developed a novel concept of endovascular thrombolysis that employs a vibrating electroactive polymer actuator. In order to predict the efficacy of thrombolysis using the developed vibrating actuator, enzyme (plasminogen activator) perfusion into a clot was analyzed by solving flow fields and species transport equations considering the fluid structure interaction. In vitro thrombolysis experiments were also performed. Computational results showed that plasminogen activator perfusion into a clot was enhanced by actuator vibration at frequencies of 1 and 5 Hz. Plasminogen activator perfusion was affected by the actuator oscillation frequencies and amplitudes that were determined by electromechanical characteristics of a polymer actuator. Computed plasminogen activator perfused volumes were compared with experimentally measured dissolved clot volumes. The computed plasminogen activator perfusion volumes with threshold concentrations of 16% of the initial plasminogen activator concentration agreed well with the in vitro experimental data. This study showed the effectiveness of actuator oscillation on thrombolysis and the validity of the computational plasminogen activator perfusion model for predicting thrombolysis in complex flow fields induced by an oscillating actuator.

European cost-effectiveness study of uPA/PAI-1 biomarkers to guide adjuvant chemotherapy decisions in breast cancer.

PubMed

Marguet, Sophie; Mazouni, Chafika; Ramaekers, Bram L T; Dunant, Ariane; Kates, Ronald; Jacobs, Volker R; Joore, Manuela A; Harbeck, Nadia; Bonastre, Julia

2016-08-01

This study investigated the cost effectiveness of guideline-recommended (American Society of Clinical Oncology, European Society of Medical Oncology) urokinase plasminogen activator (uPA)/plasminogen activator inhibitor-1 (PAI-1) biomarkers to guide adjuvant chemotherapy decisions for hormone receptor-positive, node-negative early breast cancer patients at intermediate risk of relapse, in France, Germany, and The Netherlands. uPA/PAI-1 testing was compared to chemotherapy for all patients and to no chemotherapy in two age-related subgroups (35-49 and 50-75 years). A partitioned survival analysis was performed using patient-level data for survival outcomes and secondary sources. Mean quality-adjusted life years (QALYs) and costs were estimated over a lifetime horizon to calculate the incremental net monetary benefit (INMB) at a willingness-to-pay of €50,000/QALY. Uncertainty was explored through bootstrap and probabilistic sensitivity analysis using 5000 replicates. In the 35-49 year age group, INMBs were negative when uPA/PAI-1 testing was compared to chemotherapy for all patients but positive when it was compared to no chemotherapy for the three countries. In the 50-75 year age group, INMBs of uPA/PAI-1 testing compared to both reference strategies were positive in the three countries, with cost-effectiveness probabilities for the uPA/PAI-1 strategy of 65%, 70%, and 59% for France, Germany, and the Netherlands, respectively, compared with chemotherapy for all patients, and 64%, 58%, and 65%, respectively, compared with no chemotherapy. uPA/PAI-1 testing could allow the selection of patients older than 50 years requiring chemotherapy in this population, but the cost effectiveness of this strategy is uncertain. Chemotherapy for all patients is the most cost-effective strategy for patients younger than 50 years. Copyright © 2016 Elsevier Ltd. All rights reserved.

Host-Specific Response to HCV Infection in the Chimeric SCID-beige/Alb-uPA Mouse Model: Role of the Innate Antiviral Immune Response

PubMed Central

Thompson, Jill C; Smith, Maria W; Yeh, Matthew M; Proll, Sean; Zhu, Lin-Fu; Gao, T. J; Kneteman, Norman M; Tyrrell, D. Lorne; Katze, Michael G

2006-01-01

The severe combined immunodeficiency disorder (SCID)-beige/albumin (Alb)-urokinase plasminogen activator (uPA) mouse containing a human-mouse chimeric liver is currently the only small animal model capable of supporting hepatitis C virus (HCV) infection. This model was utilized to characterize the host transcriptional response to HCV infection. The purpose of these studies was to investigate the genetic component of the host response to HCV infection and also to distinguish virus-induced gene expression changes from adaptive HCV-specific immune-mediated effects. Gene expression profiles from HCV-infected mice were also compared to those from HCV-infected patients. Analyses of the gene expression data demonstrate that host factors regulate the response to HCV infection, including the nature of the innate antiviral immune response. They also indicate that HCV mediates gene expression changes, including regulation of lipid metabolism genes, which have the potential to be directly cytopathic, indicating that liver pathology may not be exclusively mediated by HCV-specific adaptive immune responses. This effect appears to be inversely related to the activation of the innate antiviral immune response. In summary, the nature of the initial interferon response to HCV infection may determine the extent of viral-mediated effects on host gene expression. PMID:16789836

Chronic dietary fat intake modifies the postprandial response of hemostatic markers to a single fatty test meal.

PubMed

Delgado-Lista, Javier; Lopez-Miranda, Jose; Cortés, Begoña; Perez-Martinez, Pablo; Lozano, Aquiles; Gomez-Luna, Rafael; Gomez, Purificacion; Gomez, Maria Jose; Criado, Juan; Fuentes, Francisco; Perez-Jimenez, Francisco

2008-02-01

Hemostasis is the result of a complex equilibrium between coagulation and fibrinolysis, and the influence of different dietary models on this equilibrium is not entirely known. The objective was to compare the effects of the chronic intake of different dietary models on postprandial hemostasis. In a randomized crossover design, 20 healthy men consumed for 28 d each diets rich in monounsaturated fatty acids (MUFAs), saturated fatty acids (SFAs), and carbohydrates plus n-3 fatty acids (CHO/N3). Fasting and postprandial hemostatic factors (factor VII coagulant activity, plasminogen activator inhibitor-1, tissue-type plasminogen activator, d-dimer, and thromboxane B(2)) were measured; meal tests for the postprandial measures were based on butter, virgin olive oil, and walnuts for the SFA, MUFA, and CHO/N3 diets, respectively. There were no differences in the fasting variables after the dietary periods. After the 3 fatty meals were consumed, we observed an increase in thromboxane B(2) and d-dimer and a reduction in tissue plasminogen activator, irrespective of the dietary model. The MUFA or CHO/N3 meals lowered postprandial concentrations of factor VII coagulant activity, although the reduction was greater after the MUFA-enriched meal. The concentration of plasminogen activator inhibitor-1 was greater after the SFA meal than after the other 2 meals. The administration of a fatty meal induces a postprandial procoagulant tendency, irrespective of the type of fat consumed. However, the use of a dietary model rich in SFA creates a more procoagulant environment than does a model that includes MUFA or CHO/N3 as the source of fatty acids.

Keeping the blood flowing—plasminogen activator genes and feeding behavior in vampire bats

NASA Astrophysics Data System (ADS)

Tellgren-Roth, Åsa; Dittmar, Katharina; Massey, Steven E.; Kemi, Cecilia; Tellgren-Roth, Christian; Savolainen, Peter; Lyons, Leslie A.; Liberles, David A.

2009-01-01

The blood feeding vampire bats emerged from New World leaf-nosed bats that fed on fruit and insects. Plasminogen activator, a serine protease that regulates blood coagulation, is known to be expressed in the saliva of Desmodus rotundus (common vampire bat) and is thought to be a key enzyme for the emergence of blood feeding in vampire bats. To better understand the evolution of this biological function, we studied the plasminogen activator (PA) genes from all vampire bat species in light of their feeding transition to bird and subsequently mammalian blood. We include the rare species Diphylla ecaudata and Diaemus youngi, where plasminogen activator had not previously been studied and demonstrate that PA gene duplication observed in Desmodus is not essential to the vampire phenotype, but relates to the emergence of predominant mammalian blood feeding in this species. Plasminogen activator has evolved through gene duplication, domain loss, and sequence evolution leading to change in fibrin-specificity and susceptibility to plasminogen activator inhibitor-1. Before undertaking this study, only the four plasminogen activator isoforms from Desmodus were known. The evolution of vampire bat plasminogen activators can now be linked phylogenetically to the transition in feeding behavior among vampire bat species from bird to mammalian blood.

The association between the 4G/5G polymorphism in the promoter of the plasminogen activator inhibitor-1 gene and extension of postsurgical calf vein thrombosis.

PubMed

Ferrara, Filippo; Meli, Francesco; Raimondi, Francesco; Montalto, Salvatore; Cospite, Valentina; Novo, Giuseppina; Novo, Salvatore

2013-04-01

The objective of this study was to evaluate whether the presence of a plasminogen activator inhibitor type 1 (PAI-1) promoter polymorphism 4G/5G could significantly influence the proximal extension of vein thrombosis in spite of anticoagulant treatment in patients with calf vein thrombosis (CVT) following orthopaedic, urological and abdominal surgery. We studied 168 patients with CVT, who had undergone orthopaedic, urological and abdominal surgery, subdivided as follows: first, 50 patients with thrombosis progression; second, 118 patients without thrombosis progression. The 4G/5G polymorphism of the plasminogen activator inhibitor 1 was evaluated in all patients and in 70 healthy matched controls. We also studied PAI-1 activity in plasma. The presence of 4G/5G genotype was significantly increased in the group of patients with the extension of thrombotic lesions and was associated with an increase in CVT extension risk (odds ratio adjusted for sex 2.692; 95% confidence interval 1.302-4.702). Moreover, we observed a significant increase of PAI-1 plasma activity in patients with extension of thrombotic lesion vs. patients without extension (P=0.0001). Patients with 4G/5G genotype in the promoter of the plasminogen activator inhibitor - 1 gene present a higher risk of extension of thrombotic lesions.

Identification of differential pathways in papillary thyroid carcinoma utilizing pathway co-expression analysis.

PubMed

Qiu, Wei-Hai; Chen, Gui-Yan; Cui, Lu; Zhang, Ting-Ming; Wei, Feng; Yang, Yong

2016-01-01

To identify differential pathways between papillary thyroid carcinoma (PTC) patients and normal controls utilizing a novel method which combined pathway with co-expression network. The proposed method included three steps. In the first step, we conducted pretreatments for background pathways and gained representative pathways in PTC. Subsequently, a co-expression network for representative pathways was constructed using empirical Bayes (EB) approach to assign a weight value for each pathway. Finally, random model was extracted to set the thresholds of identifying differential pathways. We obtained 1267 representative pathways and their weight values based on the co-expressed pathway network, and then by meeting the criterion (Weight > 0.0296), 87 differential pathways in total across PTC patients and normal controls were identified. The top three ranked differential pathways were CREB phosphorylation, attachment of GPI anchor to urokinase plasminogen activator receptor (uPAR) and loss of function of SMAD2/3 in cancer. In conclusion, we successfully identified differential pathways (such as CREB phosphorylation, attachment of GPI anchor to uPAR and post-translational modification: synthesis of GPI-anchored proteins) for PTC using the proposed pathway co-expression method, and these pathways might be potential biomarkers for target therapy and detection of PTC.

Diabetes-Induced Superoxide Anion and Breakdown of the Blood-Retinal Barrier: Role of the VEGF/uPAR Pathway

PubMed Central

El-Remessy, Azza B.; Franklin, Telina; Ghaley, Nagla; Yang, Jinling; Brands, Michael W.; Caldwell, Ruth B.; Behzadian, Mohamed Ali

2013-01-01

Diabetes-induced breakdown of the blood-retinal barrier (BRB) has been linked to hyperglycemia-induced expression of vascular endothelial growth factor (VEGF) and is likely mediated by an increase in oxidative stress. We have shown that VEGF increases permeability of retinal endothelial cells (REC) by inducing expression of urokinase plasminogen activator receptor (uPAR). The purpose of this study was to define the role of superoxide anion in VEGF/uPAR expression and BRB breakdown in diabetes. Studies were performed in streptozotocin diabetic rats and mice and high glucose (HG) treated REC. The superoxide dismutase (SOD) mimetic tempol blocked diabetes-induced permeability and uPAR expression in rats and the cell permeable SOD inhibited HG-induced expression of uPAR and VEGF in REC. Inhibiting VEGFR blocked HG-induced expression of VEGF and uPAR and GSK-3β phosphorylation in REC. HG caused β-catenin translocation from the plasma membrane into the cytosol and nucleus. Treatment with HG-conditioned media increased REC paracellular permeability that was blocked by anti-uPA or anti-uPAR antibodies. Moreover, deletion of uPAR blocked diabetes-induced BRB breakdown and activation of MMP-9 in mice. Together, these data indicate that diabetes-induced oxidative stress triggers BRB breakdown by a mechanism involving uPAR expression through VEGF-induced activation of the GSK3β/β-catenin signaling pathway. PMID:23951261

Property of hepatitis B virus replication in Tupaia belangeri hepatocytes.

PubMed

Sanada, Takahiro; Tsukiyama-Kohara, Kyoko; Yamamoto, Naoki; Ezzikouri, Sayeh; Benjelloun, Soumaya; Murakami, Shuko; Tanaka, Yasuhito; Tateno, Chise; Kohara, Michinori

2016-01-08

The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3-6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10(5) copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10(4)-10(6) copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10(3) copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Prognosis in adenocarcinomas of lower oesophagus, gastro-oesophageal junction and cardia evaluated by uPAR-immunohistochemistry.

PubMed

Laerum, Ole Didrik; Ovrebo, Kjell; Skarstein, Arne; Christensen, Ib Jarle; Alpízar-Alpízar, Warner; Helgeland, Lars; Danø, Keld; Nielsen, Boye Schnack; Illemann, Martin

2012-08-01

Adenocarcinomas of lower oesophagus, gastro-oesophageal junction and cardia in humans are highly invasive tumours with poor prognosis. The localisation of urokinase-type plasminogen activator receptor (uPAR) was determined in 66 patients; 60 with adenocarcinomas and six cases with Barrett's oesophagus. uPAR was expressed in nearly all cases of invasive adenocarcinomas by populations of cancer cells, macrophages and myofibroblasts at both the invasion front and the tumour core. In areas with high-grade dysplasia or with Barrett's metaplasia adjacent to the tumour tissue, no uPAR-immunoreactivity was found. High local expression of uPAR, therefore, appears to be a characteristic marker for invasive behaviour in this tumour, suggesting that uPAR's contribution to matrix degradation during invasive growth is a late event in carcinogenesis. Using a scoring system for semiquantitative estimation of uPAR-positivity on immmunohistochemically stained specimens, a significant association was found between poor overall survival and high uPAR-score for cancer cells in the tumour core and for macrophages peripherally at the tumour invasion zone. In multivariate analysis, these two uPAR-scores were confirmed as highly significant prognostic parameters independent of Tumour, Node, Metastasis (TNM)-stage and World Health Organization (WHO) classification. The proteolytic action of these malignant and nonmalignant accessory cells thus seemed to follow two main patterns: one dominated by uPAR positive cancer cells and one by uPAR-positive macrophages. Scoring of uPAR-positivity might be a useful parameter for onset of invasion and prognosis in these adenocarcinomas. Copyright © 2011 UICC.

Protecting Podocytes: A Key Target for Therapy of Focal Segmental Glomerulosclerosis.

PubMed

Campbell, Kirk N; Tumlin, James A

2018-05-31

Focal segmental glomerulosclerosis (FSGS) is a histologic pattern of injury demonstrated by renal biopsy that can arise from a diverse range of causes and mechanisms. It has an estimated incidence of 7 per 1 million and is the most common primary glomerular disorder leading to end-stage renal disease in the United States. This review focuses on damage to the podocyte and the consequences of this injury in patients with FSGS, the genetics of FSGS, and approaches to treatment with a focus on the effects on podocytes. The podocyte is central to the glomerular filtration barrier and is particularly vulnerable because of its highly differentiated post-mitotic phenotype. The progressive structural changes involved in the pathology of FSGS include podocyte foot process effacement, death of podocytes and exposure of the glomerular basement membrane, filtration of nonspecific plasma proteins, expansion of capillaries, misdirected filtration at points of synechiae, and mesangial matrix proliferation. Although damage to and death of podocytes can result from single-gene disorders, evidence also suggests a role for soluble factors, such as soluble urokinase-type plasminogen activator receptor, cardiotrophin-like cytokine-1, and anti-CD40 antibodies, that promote FSGS recurrence post transplant. Several classes of medications, including corticosteroids, calcineurin inhibitors, endothelin receptor antagonists, adrenocorticotropic hormone, and rituximab, have been shown to be effective for the treatment of FSGS and have been demonstrated to have significant protective effects on podocytes. Key Messages: Greater understanding of podocyte biology is essential to the identification of new treatment targets and medications for the management of patients with FSGS. © 2018 S. Karger AG, Basel.

Virtual screening using molecular simulations.

PubMed

Yang, Tianyi; Wu, Johnny C; Yan, Chunli; Wang, Yuanfeng; Luo, Ray; Gonzales, Michael B; Dalby, Kevin N; Ren, Pengyu

2011-06-01

Effective virtual screening relies on our ability to make accurate prediction of protein-ligand binding, which remains a great challenge. In this work, utilizing the molecular-mechanics Poisson-Boltzmann (or Generalized Born) surface area approach, we have evaluated the binding affinity of a set of 156 ligands to seven families of proteins, trypsin β, thrombin α, cyclin-dependent kinase (CDK), cAMP-dependent kinase (PKA), urokinase-type plasminogen activator, β-glucosidase A, and coagulation factor Xa. The effect of protein dielectric constant in the implicit-solvent model on the binding free energy calculation is shown to be important. The statistical correlations between the binding energy calculated from the implicit-solvent approach and experimental free energy are in the range of 0.56-0.79 across all the families. This performance is better than that of typical docking programs especially given that the latter is directly trained using known binding data whereas the molecular mechanics is based on general physical parameters. Estimation of entropic contribution remains the barrier to accurate free energy calculation. We show that the traditional rigid rotor harmonic oscillator approximation is unable to improve the binding free energy prediction. Inclusion of conformational restriction seems to be promising but requires further investigation. On the other hand, our preliminary study suggests that implicit-solvent based alchemical perturbation, which offers explicit sampling of configuration entropy, can be a viable approach to significantly improve the prediction of binding free energy. Overall, the molecular mechanics approach has the potential for medium to high-throughput computational drug discovery. Copyright © 2011 Wiley-Liss, Inc.

Lanthanide-doped NaScF4 nanoprobes: crystal structure, optical spectroscopy and biodetection

NASA Astrophysics Data System (ADS)

Ai, Yu; Tu, Datao; Zheng, Wei; Liu, Yongsheng; Kong, Jintao; Hu, Ping; Chen, Zhuo; Huang, Mingdong; Chen, Xueyuan

2013-06-01

Trivalent lanthanide ions (Ln3+)-doped inorganic nanoparticles (NPs) as potential luminescent bioprobes have been attracting tremendous interest because of their unique upconversion (UC) and downconversion (DC) luminescence properties. NaScF4, as an important host material, has been rarely reported and its crystal structure remains unclear. Herein, based on the single crystal X-ray diffraction, the space group of NaScF4 crystals was determined to be P31 containing multiple sites of Sc3+ with crystallographic site symmetry of C1, which was verified by high-resolution photoluminescence spectroscopy of Eu3+ at low temperature (10 K). Furthermore, monodisperse and size-controllable NaScF4:Ln3+ NPs were synthesized via a facile thermal decomposition method. The biotinylated NaScF4:Er3+/Yb3+ NPs were demonstrated for their applications as a heterogeneous UC luminescence bioprobe to detect avidin with a detection limit of 180 pM. After bioconjugation with amino-terminal fragment (ATF) of urokinase plasminogen activator (uPA), NaScF4:Ln3+ NPs also exhibited specific recognition of cancer cells overexpressed with uPA receptor (uPAR, an important marker of tumor biology and metastasis), showing great potentials in tumor-targeted bioimaging.Trivalent lanthanide ions (Ln3+)-doped inorganic nanoparticles (NPs) as potential luminescent bioprobes have been attracting tremendous interest because of their unique upconversion (UC) and downconversion (DC) luminescence properties. NaScF4, as an important host material, has been rarely reported and its crystal structure remains unclear. Herein, based on the single crystal X-ray diffraction, the space group of NaScF4 crystals was determined to be P31 containing multiple sites of Sc3+ with crystallographic site symmetry of C1, which was verified by high-resolution photoluminescence spectroscopy of Eu3+ at low temperature (10 K). Furthermore, monodisperse and size-controllable NaScF4:Ln3+ NPs were synthesized via a facile thermal decomposition method. The biotinylated NaScF4:Er3+/Yb3+ NPs were demonstrated for their applications as a heterogeneous UC luminescence bioprobe to detect avidin with a detection limit of 180 pM. After bioconjugation with amino-terminal fragment (ATF) of urokinase plasminogen activator (uPA), NaScF4:Ln3+ NPs also exhibited specific recognition of cancer cells overexpressed with uPA receptor (uPAR, an important marker of tumor biology and metastasis), showing great potentials in tumor-targeted bioimaging. Electronic supplementary information (ESI) available: Crystallographic data (CCDC 931481) in CIF format. EDX analysis of NaScF4:Er3+/Yb3+ NPs. 10 K PL excitation spectra of NaScF4:Eu3+ microcrystals. Selected bond lengths and angles for NaScF4 crystals. Atomic coordinates and equivalent isotropic displacement parameters for NaScF4 crystals. UC quantum yield data of NaScF4:Er3+/Yb3+ NPs. See DOI: 10.1039/c3nr01529g

Conditionally activating optical contrast agent with enhanced sensitivity via gold nanoparticle plasmon energy transfer: feasibility study.

PubMed

Kang, Kyung Aih; Wang, Jianting

2014-12-07

Molecular sensing/imaging utilizing fluorophores has been one of the most frequently used techniques in biomedical research. As for any molecular imaging techniques, fluorescence mediated sensing always seeks for greater specificity and sensitivity. Since fluorophores emit fluorescence while their electron energy state changes, manipulating the local electromagnetic field around the fluorophores may be a way to enhance the specificity and sensitivity. Gold nanoparticles (GNPs) are known to form a very strong electromagnetic field on their surface [i.e., surface plasmon field (SPF)], upon receiving photonic energy. The level of fluorescence change by GNP-SPF may range from complete quenching to extensive enhancement, depending upon the SPF strength, excitation and emission wavelengths, and quantum yield of the fluorophore. Here, we report a novel design that utilizes BOTH fluorescence quenching and enhancement abilities of the GNP in one single nano-entity, providing high specificity and sensitivity. The construct utilizes a specially designed molecular dual-spacer that places the fluorphore at the location with an appropriate GNP-SFP strength before and after exposed to the biomarker. A model system to test the concept was an optical signal mediator activated by urokinase-type plasminogen activator (uPA; breast cancer secreting enzyme). The resulting contrast agent shows less than 10% of the natural fluorescence but, in the presence of uPA, its fluorescence emission is triggered and emits its fluorescence approximately twice of the natural form. This study demonstrated that our novel design of an optical contrast agent can be conditionally activated with enhanced sensitivity, using both quenching and enhancement phenomena of fluorophores in the electromagnetic field of the appropriate strengths (in this case, locally generated by the GNP-SPF). This entity is similar to molecular beacon in terms of specificity but with greater sensitivity. In addition, it is not restricted to only DNA or RNA sensing but for any designs that cause the change in the distance between the fluorophore and GNP, upon the time of encountering biomarker of interest.

The Adult Livers of Immunodeficient Mice Support Human Hematopoiesis: Evidence for a Hepatic Mast Cell Population that Develops Early in Human Ontogeny

PubMed Central

Muench, Marcus O.; Beyer, Ashley I.; Fomin, Marina E.; Thakker, Rahul; Mulvaney, Usha S.; Nakamura, Masato; Suemizu, Hiroshi; Bárcena, Alicia

2014-01-01

The liver plays a vital role in hematopoiesis during mammalian prenatal development but its hematopoietic output declines during the perinatal period. Nonetheless, hepatic hematopoiesis is believed to persist into adulthood. We sought to model human adult-liver hematopoiesis by transplantation of fetal and neonatal hematopoietic stem cells (HSCs) into adult immunodeficient mice. Livers were found to be engrafted with human cells consisting primarily of monocytes and B-cells with lesser contributions by erythrocytes, T-cells, NK-cells and mast-cells. A resident population of CD117++CD203c+ mast cells was also documented in human midgestation liver, indicating that these cells comprise part of the liver's resident immune cell repertoire throughout human ontogeny. The murine liver was shown to support human multilineage hematopoiesis up to 321 days after transplant. Evidence of murine hepatic hematopoiesis was also found in common mouse strains as old as 2 years. Human HSC engraftment of the murine liver was demonstrated by detection of high proliferative-potential colony-forming cells in clonal cultures, observation of CD38−CD34++ and CD133+CD34++ cells by flow cytometry, and hematopoietic reconstitution of secondary transplant recipients of chimeric liver cells. Additionally, chimeric mice with both hematopoietic and endothelial reconstitution were generated by intrasplenic injection of immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA) transgene. In conclusion, the murine liver is shown to be a hematopoietic organ throughout adult life that can also support human hematopoiesis in severely immunodeficient strains. Further humanization of the murine liver can be achieved in mice harboring an uPA transgene, which support engraftment of non-hematopoietic cells types. Thus, offering a model system to study the interaction of diverse human liver cell types that regulate hematopoiesis and immune function in the liver. PMID:24819392

suPAR level is associated with myocardial impairment assessed with advanced echocardiography in patients with type 1 diabetes with normal ejection fraction and without known heart disease or end-stage renal disease.

PubMed

Theilade, Simone; Rossing, Peter; Eugen-Olsen, Jesper; Jensen, Jan S; Jensen, Magnus T

2016-06-01

Heart disease is a common fatal diabetes-related complication. Early detection of patients at particular risk of heart disease is of prime importance. Soluble urokinase plasminogen activator receptor (suPAR) is a novel biomarker for development of cardiovascular disease. We investigate if suPAR is associated with early myocardial impairment assessed with advanced echocardiographic methods. In an observational study on 318 patients with type 1 diabetes without known heart disease and with normal left ventricular ejection fraction (LVEF) (biplane LVEF >45%), we performed conventional, tissue Doppler and speckle tracking echocardiography, and measured plasma suPAR levels. Associations between myocardial function and suPAR levels were studied in adjusted models including significant covariates. Patients were 55±12 years (mean±s.d.) and 160 (50%) males. Median (interquartile range) suPAR was 3.4 (1.7) ng/mL and LVEF was 58±5%. suPAR levels were not associated with LVEF (P=0.11). In adjusted models, higher suPAR levels were independently associated with both impaired systolic function assessed with global longitudinal strain (GLS) and tissue velocity s', and with impaired diastolic measures a' and e'/a' (all P=0.034). In multivariable analysis including cardiovascular risk factors and both systolic and diastolic measures (GLS and e'/a'), both remained independently associated with suPAR levels (P=0.012). In patients with type 1 diabetes with normal LVEF and without known heart disease, suPAR is associated with early systolic and diastolic myocardial impairment. Our study implies that both suPAR and advanced echocardiography are useful diagnostic tools for identifying patients with diabetes at risk of future clinical heart disease, suited for intensified medical therapy. © 2016 European Society of Endocrinology.

Curcumin suppresses colon cancer cell invasion via AMPK-induced inhibition of NF-κB, uPA activator and MMP9.

PubMed

Tong, Weihua; Wang, Quan; Sun, Donghui; Suo, Jian

2016-11-01

Curcumin, an active nontoxic ingredient of turmeric, possesses potent anti-inflammatory, antioxidant and anti-cancer properties; however, the molecular mechanisms of curcumin are not fully understood. The transcription factor nuclear factor-κB (NF-κB) is key in cellular processes, and the expression/activation of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9) are crucial for cell invasion. The present study investigated the hypothesis that curcumin inhibits colon cancer cell invasion by modulating NF-κB-mediated expression and activation of uPA and MMP9. Human colon cancer SW480 and LoVo cells were treated with various concentrations of curcumin. Curcumin was demonstrated to dose-dependently inhibit the adhesion and proliferation ability of LoVo and SW480 cells using Transwell and MTT assays, respectively. In addition, curcumin activated 5' AMP-activated protein kinase (AMPK) and suppressed p65 NF-κB phosphorylation, as shown by western blot analysis. Compound C, a potent AMPK inhibitor, abolished curcumin-induced inhibition of NF-κB, uPA and MMP9, suggesting that AMPK activation is responsible for curcumin-mediated NF-κB, uPA and MMP9 inhibition. The binding activity of NF-κB to DNA was examined and western blotting and quantitative polymerase reaction was performed to detect the effect of curcumin on the expression of uPA and MMP9. The present results revealed that curcumin significantly decreased the expression of uPA and MMP9 and NF-κB DNA binding activity. Furthermore, curcumin decreased the level of the p65 subunit of NF-κB binding to the promoter of the gene encoding uPA and MMP9, which suppressed transcriptional activation of uPA and MMP9. Overall, the present data suggest that curcumin inhibits colon cancer cell invasion via AMPK activation and subsequent inhibition of p65 NF-κB, uPA and MMP9. The therapeutic potential of curcumin for colon cancer metastasis required additional study.

Tissue plasminogen activator (tPA) and matrix metalloproteinases in the pathogenesis of stroke: therapeutic strategies.

PubMed

Adibhatla, Rao Muralikrishna; Hatcher, James F

2008-06-01

Today there exists only one FDA-approved treatment for ischemic stroke; i.e., the serine protease tissue-type plasminogen activator (tPA). In the aftermath of the failed stroke clinical trials with the nitrone spin trap/radical scavenger, NXY-059, a number of articles raised the question: are we doing the right thing? Is the animal research truly translational in identifying new agents for stroke treatment? This review summarizes the current state of affairs with plasminogen activators in thrombolytic therapy. In addition to therapeutic value, potential side effects of tPA also exist that aggravate stroke injury and offset the benefits provided by reperfusion of the occluded artery. Thus, combinational options (ultrasound alone or with microspheres/nanobubbles, mechanical dissociation of clot, activated protein C (APC), plasminogen activator inhibitor-1 (PAI-1), neuroserpin and CDP-choline) that could offset tPA toxic side effects and improve efficacy are also discussed here. Desmoteplase, a plasminogen activator derived from the saliva of Desmodus rotundus vampire bat, antagonizes vascular tPA-induced neurotoxicity by competitively binding to low-density lipoprotein related-receptors (LPR) at the blood-brain barrier (BBB) interface, minimizing the tPA uptake into brain parenchyma. tPA can also activate matrix metalloproteinases (MMPs), a family of endopeptidases comprised of 24 mammalian enzymes that primarily catalyze the turnover and degradation of the extracellular matrix (ECM). MMPs have been implicated in BBB breakdown and neuronal injury in the early times after stroke, but also contribute to vascular remodeling, angiogenesis, neurogenesis and axonal regeneration during the later repair phase after stroke. tPA, directly or by activation of MMP-9, could have beneficial effects on recovery after stroke by promoting neurovascular repair through vascular endothelial growth factor (VEGF). However, any treatment regimen directed at MMPs must consider their pleiotropic nature and the likelihood of either beneficial or detrimental effects that might depend on the timing of the treatment in relation to the stage of brain injury.

Effects of the tat and nef gene products of human immunodeficiency virus type 1 (HIV-1) on transcription controlled by the HIV-1 long terminal repeat and on cell growth in macrophages.

PubMed Central

Murphy, K M; Sweet, M J; Ross, I L; Hume, D A

1993-01-01

The RAW264 murine macrophage cell line was used as a model to examine the role of the tat and nef gene products in the transcription regulation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in macrophages. Contrary to claims that the activity of the HIV-1 LTR responds poorly in rodent cells to trans activation by the viral tat gene product, cotransfection of RAW264 cells with a tat expression plasmid in transient transfection assays caused a > 20-fold increase in reporter gene expression that was inhibited by mutations in the TAR region. RAW264 cells stably transfected with the tat plasmid displayed similarly elevated HIV-1 LTR-driven reporter gene activity. By contrast to previous reports indicating a negative role for nef in HIV transcription, cotransfection of RAW264 cells with a nef expression plasmid trans activated the HIV-1 LTR driving either a chloramphenicol acetyltransferase or a luciferase reporter gene. The action of nef was specific to the LTR, as expression of nef had no effect on the activity of the simian virus 40, c-fms, urokinase plasminogen activator, or type 5 acid phosphatase promoter. trans-activating activity was also manifested by a frameshift mutant expressing only the first 35 amino acids of the protein. The effects of nef were multiplicative with those of tat gene product and occurred even in the presence of bacterial lipopolysaccharide, which itself activated LTR-directed transcription. Examination of the effects of selected mutations in the LTR revealed that neither the kappa B sites in the direct repeat enhancer nor the TAR region was required as a cis-acting element in nef action. The action of nef was not species restricted; it was able to trans activate in the human monocyte-like cell line Mono Mac 6. The presence of a nef expression cassette in a neomycin phosphotransferase gene expression plasmid greatly reduced the number of G418-resistant colonies generated in stable transfection of RAW264 cells, and many of the colonies that were formed exhibited very slow growth. The frameshift mutant was also active in reducing colony generation. Given the absence of any effect of the frameshift mutation on nef function, its actions on macrophage growth and HIV transcription are discussed in terms of the role of the N-terminal 30 amino acids and of stable secondary structures in the mRNA. Images PMID:8230418

uPAR and Cathepsin B Downregulation Induces Apoptosis by Targeting Calcineurin A to BAD via Bcl-2 in Glioma

PubMed Central

Malla, Rama Rao; Gopinath, Sreelatha; Gondi, Christopher S.; Alapati, Kiranmai; Dinh, Dzung H.; Tsung, Andrew J.; Rao, Jasti S.

2011-01-01

Cathepsin B and urokinase plasminogen activator receptor (uPAR) are postulated to play key roles in glioma invasion. Calcineurin is one of the key regulators of mitochondrial-dependent apoptosis, but its mechanism is poorly understood. Hence, we studied subcellular localization of calcineurin after transcriptional downregulation of uPAR and cathepsin B in glioma. In the present study, efficient downregulation of uPAR and cathepsin B increased the translocation of calcineurin A from the mitochondria to the cytosol, decreased pBAD (S136) expression and its interaction with 14-3-3ζ, and increased the interaction of BAD with Bcl-Xl. Co-depletion of uPAR and cathepsin B induced mitochondrial translocation of BAD and caspase 3 as well as PARP activation, cytochrome c and SMAC release. These effects were inhibited by FK506 (10 μM), a specific inhibitor of calcineurin. Calcineurin A was co-localized and also co-immunoprecipitated with Bcl-2. This interaction decreased with co-depletion of uPAR and cathepsin B and also with Bcl-2 inhibitor, HA 14-1 (20 μg/mL). Altered localization and interaction of calcineurin A with Bcl-2 was also observed in vivo when uPAR and cathepsin B were downregulated. In conclusion, downregulation of uPAR and cathepsin B induced apoptosis by targeting calcineurin A to BAD via Bcl-2 in glioma. PMID:21964739

Estradiol agonists inhibit human LoVo colorectal-cancer cell proliferation and migration through p53.

PubMed

Hsu, Hsi-Hsien; Kuo, Wei-Wen; Ju, Da-Tong; Yeh, Yu-Lan; Tu, Chuan-Chou; Tsai, Ying-Lan; Shen, Chia-Yao; Chang, Sheng-Huang; Chung, Li-Chin; Huang, Chih-Yang

2014-11-28

To investigate the effects of 17β-estradiol via estrogen receptors (ER) or direct administration of ER agonists on human colorectal cancer. LoVo cells were established from the Bioresource Collection and Research Center and cultured in phenol red-free DMEM (Sigma, United States). To investigate the effects of E2 and/or ER selective agonists on cellular proliferation, LoVo colorectal cells were treated with E2 or ER-selective agonists for 24 h and 48 h and subjected to the MTT (Sigma) assay to find the concentration. And investigate the effects of E2 and/or ER selective agonists on cell used western immunoblotting to find out the diversification of signaling pathways. In order to observe motility and migration the wound healing assay and a transwell chamber (Neuro Probe) plate were tased. For a quantitative measure, we counted the number of migrating cells to the wound area post-wounding for 24 h. We further examined the cellular migration-regulating factors urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and matrix metalloproteinase (MMP)-9 in human LoVo cells so gelatin zymography that we used and gelatinolytic activity was visualized by Coomassie blue staining. And these results are presented as means ± SE, and statistical comparisons were made using Student's t-test. The structure was first compared with E2 and ER agonists. We then treated the LoVo cells with E2 and ER agonists (10(-8) mol/L) for 24 h and 48 h and subsequently measured the cell viability using MTT assay. Our results showed that treatment with 17β-estradiol and/or ER agonists in human LoVo colorectal cancer cells activated p53 and then up-regulated p21 and p27 protein levels, subsequently inhibiting the downstream target gene, cyclin D1, which regulates cell proliferation. Taken together, our findings demonstrate the anti-tumorigenesis effects of 17β-estradiol and/or ER agonists and suggest that these compounds may prove to be a potential alternative therapy in the treatment of human colorectal cancer. These results demonstrate that 17β-estradiol and/or ER agonists downregulate migration-related proteins through the p53 signaling pathway in human LoVo colorectal cancer cells. These findings suggest that p53 plays a critical role in the 17β-estradiol and/or ER agonist-mediated protective activity against colorectal cancer progression. In addition, 17β-estradiol and/or ER agonists dramatically inhibited cell migration and reduced the expression of u-PA, t-PA and MMP-9 as well as MMP-2/9 activity in LoVo cells, which regulate cell metastasis. Moreover, we observed that pretreatment with a p53 inhibitor significantly blocked the anti-migration effects of E2 and/or ER agonists on LoVo cells. That E2 and/or ER agonists may impair LoVo cell migration by modulating migration-related factors via the p53 tumor suppressor gene. Direct ER treatment may prove to be an attractive alternative therapy in the treatment of human colorectal tumors in the future.

Surface-Expressed Enolase Contributes to the Pathogenesis of Clinical Isolate SSU of Aeromonas hydrophila▿

PubMed Central

Sha, Jian; Erova, Tatiana E.; Alyea, Rebecca A.; Wang, Shaofei; Olano, Juan P.; Pancholi, Vijay; Chopra, Ashok K.

2009-01-01

In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU of Aeromonas hydrophila bound to human plasminogen and facilitated the latter's tissue-type plasminogen activator-mediated activation to plasmin. The bacterial surface-bound plasmin was more resistant to the action of its specific physiological inhibitor, the antiprotease α2-antiplasmin. We found that immunization of mice with purified recombinant enolase significantly protected the animals against a lethal challenge dose of wild-type (WT) A. hydrophila. Minimal histological changes were noted in organs from mice immunized with enolase and then challenged with WT bacteria compared to severe pathological changes found in the infected and nonimmunized group of animals. This correlated with the smaller bacterial load of WT bacteria in the livers and spleens of enolase-immunized mice than that found in the nonimmunized controls. We also showed that the enolase gene could potentially be important for the viability of A. hydrophila SSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied in trans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase in A. hydrophila SSU; the mutated forms of enolase were hyperexpressed in Escherichia coli, and the proteins were purified. Our results indicated that lysine residues at positions 420 and 427 of enolase were crucial in plasminogen-binding activity. We also identified a stretch of amino acid residues (252FYDAEKKEY260) in the A. hydrophila SSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis of A. hydrophila SSU infections and of any gram-negative bacteria in general. PMID:19270100

DOE Office of Scientific and Technical Information (OSTI.GOV)

Busch, Susann; Renaud, Stephen J.; Schleussner, Ekkehard

The intracellular signaling molecule mammalian target of rapamycin (mTOR) is essential for cell growth and proliferation. It is involved in mouse embryogenesis, murine trophoblast outgrowth and linked to tumor cell invasiveness. In order to assess the role of mTOR in human trophoblast invasion we analyzed the in vitro invasiveness of HTR-8/SVneo immortalized first-trimester trophoblast cells in conjunction with enzyme secretion upon mTOR inhibition and knockdown of mTOR protein expression. Additionally, we also tested the capability of mTOR to trigger signal transducer and activator of transcription (STAT)-3 by its phosphorylation status. Rapamycin inhibited mTOR kinase activity as demonstrated with a lowermore » phosphorylation level of the mTOR substrate p70 S6 kinase (S6K). With the use of rapamycin and siRNA-mediated mTOR knockdown we could show that cell proliferation, invasion and secretion of matrix-metalloproteinases (MMP)-2 and -9, urokinase-like plasminogen activator (uPA) and its major physiological uPA inhibitor (PAI)-1 were inhibited. While tyrosine phosphorylation of STAT3 was unaffected by mTOR inhibition and knockdown, serine phosphorylation was diminished. We conclude that mTOR signaling is one major mechanism in a tightly regulated network of intracellular signal pathways including the JAK/STAT system to regulate invasion in human trophoblast cells by secretion of enzymes that remodel the extra-cellular matrix (ECM) such as MMP-2, -9, uPA and PAI-1. Dysregulation of mTOR may contribute to pregnancy-related pathologies caused through impaired trophoblast invasion.« less

Fish oil decreases matrix metalloproteinases in knee synovia of dogs with inflammatory joint disease.

PubMed

Hansen, Rodney A; Harris, Mary A; Pluhar, G Elizabeth; Motta, Tatiana; Brevard, Sean; Ogilvie, Gregory K; Fettman, Martin J; Allen, Kenneth G D

2008-02-01

This study was designed to determine whether dietary fish oil affects the expression and activity of matrix metalloproteinases (MMP), tissue inhibitors of MMP-2 (TIMP-2) and urokinase plasminogen activator (uPA) in synovial fluid from dogs with spontaneously occurring stifle (knee) instability in a single hind limb resulting from acute cranial cruciate ligament (CCL) injury. Two groups of 12 dogs were fed diets from 1 week prior to surgery on the affected knee to 56 days post-surgery. The fish oil and control diets provided 90 and 4.5 mg, respectively, of combined eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)/kg body weight per day. Plasma and synovial fluid, from both surgical and nonsurgical knee joints, were obtained at start of the diet (-7), surgery day (0) and 7, 14, 28 and 56 days post-surgery. Plasma total EPA and DHA were significantly increased, and plasma total arachidonic acid (AA) was significantly decreased by the fish oil diet. In synovial fluid from the nonsurgical knee, fish oil treatment significantly decreased proMMP-2 expression at Days 7 and 14, and proMMP-9 expression at Day 56, and uPA activity at 28 days and significantly increased TIMP-2 expression at Days 7 and 28. There were no differences in MMP expression or activity, TIMP-2 expression and uPA activity in the surgical joint synovial fluid at any time throughout the study. These results suggest that dietary fish oil may exert beneficial effects on synovial fluid MMP and TIMP-2 equilibrium in the uninjured stifle of dogs with unilateral CCL injury.

Mature and progenitor endothelial cells perform angiogenesis also under protease inhibition: the amoeboid angiogenesis.

PubMed

Chillà, Anastasia; Margheri, Francesca; Biagioni, Alessio; Del Rosso, Mario; Fibbi, Gabriella; Laurenzana, Anna

2018-04-03

Controlling vascular growth is a challenging aim for the inhibition of tumor growth and metastasis. The amoeboid and mesenchymal types of invasiveness are two modes of migration interchangeable in cancer cells: the Rac-dependent mesenchymal migration requires the activity of proteases; the Rho-ROCK-dependent amoeboid motility is protease-independent and has never been described in endothelial cells. A cocktail of physiologic inhibitors (Ph-C) of serine-proteases, metallo-proteases and cysteine-proteases, mimicking the physiological environment that cells encounter during their migration within the angiogenesis sites was used to induce amoeboid style migration of Endothelial colony forming cells (ECFCs) and mature endothelial cells (ECs). To evaluate the mesenchymal-ameboid transition RhoA and Rac1 activation assays were performed along with immunofluorescence analysis of proteins involved in cytoskeleton organization. Cell invasion was studied in Boyden chambers and Matrigel plug assay for the in vivo angiogenesis. In the present study we showed in both ECFCs and ECs, a decrease of activated Rac1 and an increase of activated RhoA upon shifting of cells to the amoeboid conditions. In presence of Ph-C inhibitors both cell lines acquired a round morphology and Matrigel invasion was greatly enhanced with respect to that observed in the absence of protease inhibition. We also observed that the urokinase-plasminogen-activator (uPAR) receptor silencing and uPAR-integrin uncoupling with the M25 peptide abolished both mesenchymal and amoeboid angiogenesis of ECFCs and ECs in vitro and in vivo, indicating a role of the uPAR-integrin-actin axis in the regulation of amoeboid angiogenesis. Furthermore, under amoeboid conditions endothelial cells seem to be indifferent to VEGF stimulation, which induces an amoeboid signaling pattern also in mesenchymal conditions. Here we first provide a data set disclosing that endothelial cells can move and differentiate into vascular structures in vitro and in vivo also in the absence of proteases activity, performing a new type of neovascularization: the "amoeboid angiogenesis". uPAR is indispensable for ECs and ECFCs to perform an efficient amoeboid angiogenesis. Therefore, uPAR silencing or the block of its integrin-interaction, together with standard treatment against VEGF, could be a possible solution for angiogenesis inhibition.

Nuclear receptors in pancreatic tumor cells.

PubMed

Damaskos, Christos; Garmpis, Nikolaos; Karatzas, Theodore; Kostakis, Ioannis D; Nikolidakis, Lampros; Kostakis, Alkiviadis; Kouraklis, Gregory

2014-12-01

This review focuses on nuclear receptors expressed in pancreatic cancer. An extensive search of articles published up to March 2013 was conducted using the MEDLINE database. The key words used were "pancreatic cancer", "molecular receptors" and "growth factors". A total of 112 articles referred to pancreatic cancer, molecular receptors and/or growth factors were included. Receptors of growth factors, such as the epithelial growth factor receptor, insulin-like growth factor-1 receptor, vascular endothelial growth factor receptor and others, such as integrin α5β1, somatostatin receptors, the death receptor 5, claudin, notch receptors, mesothelin receptors, follicle-stimulating hormone receptors, the MUC1 receptor, the adrenomedullin receptor, the farnesoid X receptor, the transferrin receptor, sigma-2 receptors, the chemokine receptor CXCR4, the urokinase plasminogen activator receptor, the ephrine A2 receptor, the GRIA3 receptor, the RON receptor and the angiotensin II receptor AT-1 are expressed in pancreatic tumor cells. These molecules are implicated in tumor growth, apoptosis, angiogenesis, metastasis etc. After identifying the molecular receptors associated with the pancreatic cancer, many more target molecules playing important roles in tumor pathophysiology and senescence-associated signal transduction in cancer cells will be identified. This may have a significant influence on diagnosis, therapy and prognosis of pancreatic cancer. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Lanthanide-doped NaScF4 nanoprobes: crystal structure, optical spectroscopy and biodetection.

PubMed

Ai, Yu; Tu, Datao; Zheng, Wei; Liu, Yongsheng; Kong, Jintao; Hu, Ping; Chen, Zhuo; Huang, Mingdong; Chen, Xueyuan

2013-07-21

Trivalent lanthanide ions (Ln(3+))-doped inorganic nanoparticles (NPs) as potential luminescent bioprobes have been attracting tremendous interest because of their unique upconversion (UC) and downconversion (DC) luminescence properties. NaScF4, as an important host material, has been rarely reported and its crystal structure remains unclear. Herein, based on the single crystal X-ray diffraction, the space group of NaScF4 crystals was determined to be P31 containing multiple sites of Sc(3+) with crystallographic site symmetry of C1, which was verified by high-resolution photoluminescence spectroscopy of Eu(3+) at low temperature (10 K). Furthermore, monodisperse and size-controllable NaScF4:Ln(3+) NPs were synthesized via a facile thermal decomposition method. The biotinylated NaScF4:Er(3+)/Yb(3+) NPs were demonstrated for their applications as a heterogeneous UC luminescence bioprobe to detect avidin with a detection limit of 180 pM. After bioconjugation with amino-terminal fragment (ATF) of urokinase plasminogen activator (uPA), NaScF4:Ln(3+) NPs also exhibited specific recognition of cancer cells overexpressed with uPA receptor (uPAR, an important marker of tumor biology and metastasis), showing great potentials in tumor-targeted bioimaging.

Identification of Ras suppressor-1 (RSU-1) as a potential breast cancer metastasis biomarker using a three-dimensional in vitro approach.

PubMed

Gkretsi, Vasiliki; Stylianou, Andreas; Louca, Maria; Stylianopoulos, Triantafyllos

2017-04-18

Breast cancer (BC) is the most common malignant disease in women, with most patients dying from metastasis to distant organs, making discovery of novel metastasis biomarkers and therapeutic targets imperative. Extracellular matrix (ECM)-related adhesion proteins as well as tumor matrix stiffness are important determinants for metastasis. As traditional two-dimensional culture does not take into account ECM stiffness, we employed 3-dimensional collagen I gels of increasing concentration and stiffness to embed BC cells of different invasiveness (MCF-7, MDA-MB-231 and MDA-MB-231-LM2) or tumor spheroids. We tested the expression of cell-ECM adhesion proteins and found that Ras Suppressor-1 (RSU-1) is significantly upregulated in increased stiffness conditions. Interestingly, RSU-1 siRNA-mediated silencing inhibited Urokinase Plasminogen Activator, and metalloproteinase-13, whereas tumor spheroids formed from RSU-1-depleted cells lost their invasive capacity in all cell lines and stiffness conditions. Kaplan-Meier survival plot analysis corroborated our findings showing that high RSU-1 expression is associated with poor prognosis for distant metastasis-free and remission-free survival in BC patients. Taken together, our results indicate the important role of RSU-1 in BC metastasis and set the foundations for its validation as potential BC metastasis marker.

Reduced endothelial activation after exercise is associated with improved HbA1c in patients with type 2 diabetes and coronary artery disease.

PubMed

Byrkjeland, Rune; Njerve, Ida U; Arnesen, Harald; Seljeflot, Ingebjørg; Solheim, Svein

2017-03-01

We have previously reported insignificant changes in HbA 1c after exercise in patients with both type 2 diabetes and coronary artery disease. In this study, we investigated the effect of exercise on endothelial function and possible associations between changes in endothelial function and HbA 1c . Patients with type 2 diabetes and coronary artery disease ( n = 137) were randomised to 12 months exercise or standard follow-up. Endothelial function was assessed by circulating biomarkers (E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, von Willebrand factor, tissue plasminogen activator antigen, asymmetric dimethylarginine and L-arginine/asymmetric dimethylarginine ratio). Differences between the randomised groups were analysed by analysis of covariance and correlations by Spearman's rho or Pearson's correlation. No effect of exercise on endothelial function was demonstrated. The changes in HbA 1c in the exercise group correlated with changes in E-selectin ( r = 0.56, p < 0.001), intercellular adhesion molecule-1 ( r = 0.27, p = 0.052), vascular cell adhesion molecule-1 ( r = 0.32, p = 0.022) and tissue plasminogen activator antigen ( r = 0.35, p =  0.011). HbA 1c decreased significantly more in patients with versus without a concomitant reduction in E-selectin ( p =  0.002), intercellular adhesion molecule-1 ( p =  0.011), vascular cell adhesion molecule-1 ( p =  0.028) and tissue plasminogen activator antigen ( p =  0.009). Exercise did not affect biomarkers of endothelial function in patients with both type 2 diabetes and coronary artery disease. However, changes in biomarkers of endothelial activation correlated with changes in HbA 1c , and reduced endothelial activation was associated with improved HbA 1c after exercise.

Expression of the plasminogen activator system and the inhibitors PAI-1 and PAI-2 in posttraumatic lesions of the CNS and brain injuries following dramatic circulatory arrests: an immunohistochemical study.

PubMed

Dietzmann, K; von Bossanyi, P; Krause, D; Wittig, H; Mawrin, C; Kirches, E

2000-01-01

Plasminogen activators as inducible extracellular serine proteases are involved in a variety of processes, such as the degradation of brain structures. In regions of brain degradation, an increase in the expression of genes encoding cytokines and proteinases has recently been demonstrated. We tested the hypothesis, whether the plasminogen activator system as well as the plasminogen activator inhibitors are expressed and possibly involved in a proteolytic cascade that breaks down the extracellular matrix as a result of ischemic or posttraumatic brain destructions. To study this supposition, we investigated immunohistochemically the expression of tPA, uPA and its receptor, the plasminogen activator inhibitors PAI-1 and PAI-2, tetranectin as well as the laminin breakdown as an event of secondary brain injury. Brain tissue from 21 autopsy cases with severe brain injuries, material from 14 ischemic infarcts and 11 controls with acute hypoxia were used. All components of the plasminogen activator system studied were over-expressed immunohistochemically in reactive astrocytes, microglia and endothelial cells around the lesion zone. Tetranectin showed an analogous distribution to the plasminogen activator system. A reduced immunoreactivity of laminin within the identical region of destruction was detected concomitant with laminin remnants in perivascular macrophages, so that a remarkable role of the plasmin cascade in the degradation of extracellular matrix proteins in the brain is taken into consideration.

Gambogic acid-loaded magnetic Fe(3)O(4) nanoparticles inhibit Panc-1 pancreatic cancer cell proliferation and migration by inactivating transcription factor ETS1.

PubMed

Wang, Cailian; Zhang, Haijun; Chen, Yan; Shi, Fangfang; Chen, Baoan

2012-01-01

E26 transformation-specific sequence-1 (ETS1) transcription factor plays important roles in both carcinogenesis and the progression of a wide range of malignancies. Aberrant ETS1 expression correlates with aggressive tumor behavior and a poorer prognosis in patients with various malignancies. The aim of the current study was to evaluate the efficacy of a drug delivery system utilizing gambogic acid-loaded magnetic Fe(3)O(4) nanoparticles (GA-MNP-Fe(3)O(4)) on the suppression of ETS1-mediated cell proliferation and migration in Panc-1 pancreatic cancer cells. The effects caused by GA-MNP-Fe(3)O(4) on the proliferation of Panc-1 pancreatic cancer cells were evaluated using a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay while inhibition of tumor cell migration was investigated in a scratch assay. The expressions of ETS1, cyclin D1, urokinase-type plasminogen activator (u-PA), and VEGF (vascular endothelial growth factor) were examined by Western blot to elucidate the possible mechanisms involved. In Panc-1 pancreatic cancer cells, we observed that application of GA-MNP-Fe(3)O(4) was able to suppress cancer cell proliferation and prevent cells from migrating effectively. After treatment, Panc-1 pancreatic cancer cells showed significantly decreased expression of ETS1, as well as its downstream target genes for cyclin D1, u-PA, and VEGF. Our novel finding reaffirmed the significance of ETS1 in the treatment of pancreatic cancer, and application of GA-MNP-Fe(3)O(4) nanoparticles targeting ETS1 should be considered as a promising contribution for better pancreatic cancer care.

A novel uPAg-KPI fusion protein inhibits the growth and invasion of human ovarian cancer cells in vitro.

PubMed

Zhao, Li-Ping; Xu, Tian-Min; Kan, Mu-Jie; Xiao, Ye-Chen; Cui, Man-Hua

2016-05-01

Urokinase-type plasminogen activator (uPA) acts by breaking down the basement membrane and is involved in cell proliferation, migration and invasion. These actions are mediated by binding to the uPA receptor (uPAR) via its growth factor domain (GFD). The present study evaluated the effects of uPAg-KPI, a fusion protein of uPA-GFD and a kunitz protease inhibitor (KPI) domain that is present in the amyloid β-protein precursor. Using SKOV-3 cells, an ovarian cancer cell line, we examined cell viability, migration, invasion and also protein expression. Furthermore, we examined wound healing, and migration and invasion using a Transwell assay. Our data showed that uPAg-KPI treatment reduced the viability of ovarian cancer SKOV-3 cells in both a concentration and time-dependent manner by arresting tumor cells at G1/G0 phase of the cell cycle. The IC50 of uPAg-KPI was 0.5 µg/µl after 48 h treatment. At this concentration, uPAg-KPI also inhibited tumor cell colony formation, wound closure, as well as cell migration and invasion capacity. At the protein level, western blot analysis demonstrated that uPAg-KPI exerted no significant effect on the expression of total extracellular signal-regulated kinase (ERK)1/ERK2 and AKT, whereas it suppressed levels of phosphorylated ERK1/ERK2 and AKT. Thus, we suggest that this novel uPAg-KPI fusion protein reduced cell viability, colony formation, wound healing and the invasive ability of human ovarian cancer SKOV-3 cells in vitro by regulating ERK and AKT signaling. Further studies using other cell lines will confirm these findings.

A novel uPAg-KPI fusion protein inhibits the growth and invasion of human ovarian cancer cells in vitro

PubMed Central

ZHAO, LI-PING; XU, TIAN-MIN; KAN, MU-JIE; XIAO, YE-CHEN; CUI, MAN-HUA

2016-01-01

Urokinase-type plasminogen activator (uPA) acts by breaking down the basement membrane and is involved in cell proliferation, migration and invasion. These actions are mediated by binding to the uPA receptor (uPAR) via its growth factor domain (GFD). The present study evaluated the effects of uPAg-KPI, a fusion protein of uPA-GFD and a kunitz protease inhibitor (KPI) domain that is present in the amyloid β-protein precursor. Using SKOV-3 cells, an ovarian cancer cell line, we examined cell viability, migration, invasion and also protein expression. Furthermore, we examined wound healing, and migration and invasion using a Transwell assay. Our data showed that uPAg-KPI treatment reduced the viability of ovarian cancer SKOV-3 cells in both a concentration and time-dependent manner by arresting tumor cells at G1/G0 phase of the cell cycle. The IC50 of uPAg-KPI was 0.5 µg/µl after 48 h treatment. At this concentration, uPAg-KPI also inhibited tumor cell colony formation, wound closure, as well as cell migration and invasion capacity. At the protein level, western blot analysis demonstrated that uPAg-KPI exerted no significant effect on the expression of total extracellular signal-regulated kinase (ERK)1/ERK2 and AKT, whereas it suppressed levels of phosphorylated ERK1/ERK2 and AKT. Thus, we suggest that this novel uPAg-KPI fusion protein reduced cell viability, colony formation, wound healing and the invasive ability of human ovarian cancer SKOV-3 cells in vitro by regulating ERK and AKT signaling. Further studies using other cell lines will confirm these findings. PMID:27035617

Elevated cell proliferation and VEGF production by high-glucose conditions in Müller cells involve XIAP

PubMed Central

Sun, Y; Wang, D; Ye, F; Hu, D-N; Liu, X; Zhang, L; Gao, L; Song, E; Zhang, D Y

2013-01-01

Purpose Müller cells have important roles in the pathogenesis of diabetic retinopathy by promoting cell proliferation and inducing the production of vascular endothelial growth factor (VEGF) under hyperglycemic conditions. The objective of this study was to determine the potential mechanism of Müller cell proliferation and VEGF production due to high-glucose conditions. Methods Primary cultured rat Müller cells were incubated with medium containing variable concentrations of glucose and/or embelin, a specific inhibitor of X-linked inhibitor of apoptosis protein (XIAP), for 72 h. The proliferation of Müller cells was assessed by the MTT assay. The expression and/or phosphorylation of 146 proteins were assessed using protein pathway array. Results High concentrations of glucose-induced Müller cell proliferation and altered expression and/or phosphorylation of 47 proteins that have been identified to have key roles in several important signaling pathways (XIAP, VEGF, HIF1α, NFκB, etc) and are involved in the regulation of cell survival, proliferation, or apoptosis. However, Müller cell alterations induced by high-glucose conditions were counteracted by the XIAP inhibitor embelin, and 26 proteins/phosphorylations (out of 47) were restored to their normal levels. Nine proteins, including NFκB p65, p-p38, tumor necrosis factor-α, urokinase-type plasminogen activator, CREB, IL-1β, HCAM, estrogen receptor-α, and p-Stat3, were involved in regulatory networks between XIAP and VEGF. Conclusions The current study suggests that XIAP may be a potential regulator that can mediate a series of pathological changes induced by high-glucose conditions in Müller cells. Therefore, embelin could be a potential agent for the prevention and treatment of diabetic retinopathy. PMID:23928877

Genetic variants in the vitamin D pathway and breast cancer disease-free survival

PubMed Central

Brewster, Abenaa M.

2013-01-01

Epidemiological studies have investigated the association between vitamin D pathway genes and breast cancer risk; however, little is known about the association between vitamin D pathway genes and breast cancer prognosis. In a retrospective cohort of 1029 patients with early-stage breast cancer, we analyzed the association between 106 tagging single nucleotide polymorphisms (SNPs) in eight vitamin D pathway genes and breast cancer disease-free survival (DFS) using Cox regression analysis adjusted for known prognostic variables. Using a false discovery rate of 10%, six intronic SNPs were significantly associated with poorer DFS: retinoid-X receptor alpha (RXRA) SNPs (rs881658, rs11185659, rs10881583, rs881657 and rs7864987) and plasminogen activator and urokinase receptor (PLAUR) SNP (rs4251864). Treatment received (no systemic therapy, hormone therapy alone or chemotherapy) was an effect modifier of the RXRA SNPs association with DFS (P < 0.05); therefore, we stratified further analysis by treatment group. Among patients who did not receive systemic therapy, RXRA SNP [rs10881583 (P = 0.02)] was associated with poorer DFS, and among patients who received chemotherapy, RXRA SNPs (rs881658, rs11185659, rs10881583, rs881657 and rs7864987) were associated with poorer DFS (P < 0.001 for all SNPs). However, RXRA SNPs: rs10881583 (P < 0.001) and rs881657 (P = 0.02) were associated with improved DFS in patients treated with hormone therapy alone. Our results suggest that SNPs in the RXRA and PLAUR genes in the vitamin D pathway may contribute to breast cancer DFS. In particular, SNPs in RXRA may predict for poorer or improved DFS in patients, according to type of systemic treatment received. If validated, these markers could be used for risk stratification of breast cancer patients. PMID:23180655

Epigenetic silencing of triple negative breast cancer hallmarks by Withaferin A.

PubMed

Szarc Vel Szic, Katarzyna; Declerck, Ken; Crans, René A J; Diddens, Jolien; Scherf, David B; Gerhäuser, Clarissa; Vanden Berghe, Wim

2017-06-20

Triple negative breast cancer (TNBC) is characterized by poor prognosis and a DNA hypomethylation profile. Withaferin A (WA) is a plant derived steroidal lactone which holds promise as a therapeutic agent for treatment of breast cancer (BC). We determined genome-wide DNA methylation changes in weakly-metastatic and aggressive, metastatic BC cell lines, following 72h treatment to a sub-cytotoxic concentration of WA. In contrast to the DNA demethylating agent 5-aza-2'-deoxycytidine (DAC), WA treatment of MDA-MB-231 cells rather tackles an epigenetic cancer network through gene-specific DNA hypermethylation of tumor promoting genes including ADAM metallopeptidase domain 8 (ADAM8), urokinase-type plasminogen activator (PLAU), tumor necrosis factor (ligand) superfamily, member 12 (TNFSF12), and genes related to detoxification (glutathione S-transferase mu 1, GSTM1), or mitochondrial metabolism (malic enzyme 3, ME3). Gene expression and pathway enrichment analysis further reveals epigenetic suppression of multiple cancer hallmarks associated with cell cycle regulation, cell death, cancer cell metabolism, cell motility and metastasis. Remarkably, DNA hypermethylation of corresponding CpG sites in PLAU, ADAM8, TNSF12, GSTM1 and ME3 genes correlates with receptor tyrosine-protein kinase erbB-2 amplification (HER2)/estrogen receptor (ESR)/progesterone receptor (PR) status in primary BC tumors. Moreover, upon comparing differentially methylated WA responsive target genes with DNA methylation changes in different clinical subtypes of breast cancer patients in the cancer genome atlas (TCGA), we found that WA silences HER2/PR/ESR-dependent gene expression programs to suppress aggressive TNBC characteristics in favor of luminal BC hallmarks, with an improved therapeutic sensitivity. In this respect, WA may represent a novel and attractive phyto-pharmaceutical for TNBC treatment.

In vitro and in vivo inhibition of proangiogenic retinal phenotype by an antisense oligonucleotide downregulating uPAR expression.

PubMed

Lulli, Matteo; Cammalleri, Maurizio; Granucci, Irene; Witort, Ewa; Bono, Silvia; Di Gesualdo, Federico; Lupia, Antonella; Loffredo, Rosa; Casini, Giovanni; Dal Monte, Massimo; Capaccioli, Sergio

2017-08-26

Neoangiogenesis is the main pathogenic event involved in a variety of retinal diseases. It has been recently demonstrated that inhibiting the urokinase-type plasminogen activator receptor (uPAR) results in reduced angiogenesis in a mouse model of oxygen-induced retinopathy (OIR), establishing uPAR as a therapeutic target in proliferative retinopathies. Here, we evaluated in cultured human retinal endothelial cells (HRECs) and in OIR mice the potential of a specific antisense oligodeoxyribonucleotide (ASO) in blocking the synthesis of uPAR and in providing antiangiogenic effects. uPAR expression in HRECs was inhibited by lipofection with the phosphorotioated 5'-CGGCGGGTGACCCATGTG-3' ASO-uPAR, complementary to the initial translation site of uPAR mRNA. Inhibition of uPAR expression via ASO-uPAR was evaluated in HRECs by analyzing VEGF-induced tube formation and migration. In addition, the well-established and reproducible murine OIR model was used to induce retinal neovascularization in vivo. OIR mice were injected intraperitoneally with ASO-uPAR and retinopathy was evaluated considering the extent of the avascular area in the central retina and neovascular tuft formation. The ASO-uPAR specifically decreased uPAR mRNA and protein levels in HRECs and mitigated VEGF-induced tube formation and cell migration. Noteworthy, in OIR mice ASO-uPAR administration reduced both the avascular area and the formation of neovascular tufts. In conclusion, although the extrapolation of these experimental findings to the clinic is not straightforward, ASO-uPAR may be considered a potential therapeutic tool for treatment of proliferative retinal diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Circulating proteins as predictors of incident heart failure in the elderly.

PubMed

Stenemo, Markus; Nowak, Christoph; Byberg, Liisa; Sundström, Johan; Giedraitis, Vilmantas; Lind, Lars; Ingelsson, Erik; Fall, Tove; Ärnlöv, Johan

2018-01-01

To identify novel risk markers for incident heart failure using proteomic profiling of 80 proteins previously associated with cardiovascular pathology. Proteomic profiling (proximity extension assay) was performed in two community-based prospective cohorts of elderly individuals without heart failure at baseline: the Prospective Investigation of the Vasculature in Uppsala Seniors [PIVUS, n = 901, median age 70.2 (interquartile range 70.0-70.3) years, 80 events]; and the Uppsala Longitudinal Study of Adult Men [ULSAM, n = 685, median age 77.8 (interquartile range 76.9-78.1) years, 90 events]. Twenty-nine proteins were associated with incident heart failure in the discovery cohort PIVUS after adjustment for age and sex, and correction for multiple testing. Eighteen associations replicated in ULSAM. In pooled analysis of both cohorts, higher levels of nine proteins were associated with incident heart failure after adjustment for established risk factors: growth differentiation factor 15 (GDF-15), T-cell immunoglobulin and mucin domain 1 (TIM-1), tumour necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2), spondin-1 (SPON1), matrix metalloproteinase-12 (MMP-12), follistatin (FS), urokinase-type plasminogen activator surface receptor (U-PAR), osteoprotegerin (OPG), and suppression of tumorigenicity 2 (ST2). Of these, GDF-15, U-PAR, MMP-12, TRAIL-R2, SPON1 and FS were associated with worsened echocardiographic left ventricular systolic function at baseline, while only TIM-1 was positively associated with worsened diastolic function (P < 0.02 for all). Proteomic profiling identified several novel associations between proteins involved in apoptosis, inflammation, matrix remodelling, and fibrinolysis with incident heart failure in elderly individuals. Our results encourage additional studies investigating the underlying mechanisms and the clinical utility of our findings. © 2017 The Authors. European Journal of Heart Failure © 2017 European Society of Cardiology.

Alzheimer's disease biomarker discovery using in silico literature mining and clinical validation

PubMed Central

2012-01-01

Background Alzheimer’s Disease (AD) is the most widespread form of dementia in the elderly but despite progress made in recent years towards a mechanistic understanding, there is still an urgent need for disease modification therapy and for early diagnostic tests. Substantial international efforts are being made to discover and validate biomarkers for AD using candidate analytes and various data-driven 'omics' approaches. Cerebrospinal fluid is in many ways the tissue of choice for biomarkers of brain disease but is limited by patient and clinician acceptability, and increasing attention is being paid to the search for blood-based biomarkers. The aim of this study was to use a novel in silico approach to discover a set of candidate biomarkers for AD. Methods We used an in silico literature mining approach to identify potential biomarkers by creating a summarized set of assertional metadata derived from relevant legacy information. We then assessed the validity of this approach using direct assays of the identified biomarkers in plasma by immunodetection methods. Results Using this in silico approach, we identified 25 biomarker candidates, at least three of which have subsequently been reported to be altered in blood or CSF from AD patients. Two further candidate biomarkers, indicated from the in silico approach, were choline acetyltransferase and urokinase-type plasminogen activator receptor. Using immunodetection, we showed that, in a large sample set, these markers are either altered in disease or correlate with MRI markers of atrophy. Conclusions These data support as a proof of concept the use of data mining and in silico analyses to derive valid biomarker candidates for AD and, by extension, for other disorders. PMID:23113945

HIF-2α Expression Regulates Sprout Formation into 3D Fibrin Matrices in Prolonged Hypoxia in Human Microvascular Endothelial Cells.

PubMed

Nauta, Tessa D; Duyndam, Monique C A; Weijers, Ester M; van Hinsbergh, Victor M W; Koolwijk, Pieter

2016-01-01

During short-term hypoxia, Hypoxia Inducible Factors (particular their subunits HIF-1α and HIF-2α) regulate the expression of many genes including the potent angiogenesis stimulator VEGF. However, in some pathological conditions chronic hypoxia occurs and is accompanied by reduced angiogenesis. We investigated the effect of prolonged hypoxia on the proliferation and sprouting ability of human microvascular endothelial cells and the involvement of the HIFs and Dll4/Notch signaling. Human microvascular endothelial cells (hMVECs), cultured at 20% oxygen for 14 days and seeded on top of 3D fibrin matrices, formed sprouts when stimulated with VEGF-A/TNFα. In contrast, hMVECs precultured at 1% oxygen for 14 days were viable and proliferative, but did not form sprouts into fibrin upon VEGF-A/TNFα stimulation at 1% oxygen. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting, whereas HIF-1α or HIF-3α by si-RNA had no effect. No involvement of Dll4/Notch pathway in the inhibitory effect on endothelial sprouting by prolonged hypoxia was found. In addition, hypoxia decreased the production of urokinase-type plasminogen activator (uPA), needed for migration and invasion, without a significant effect on its inhibitor PAI-1. This was independent of HIF-2α, as si-HIF-2α did not counteract uPA reduction. Prolonged culturing of hMVECs at 1% oxygen inhibited endothelial sprouting into fibrin. Two independent mechanisms contribute. Silencing of HIF-2α with si-RNA partially restored the inhibition of endothelial sprouting pointing to a HIF-2α-dependent mechanism. In addition, reduction of uPA contributed to reduced endothelial tube formation in a fibrin matrix during prolonged hypoxia.

Proteolysis of plasminogen activator inhibitor-1 by Yersinia pestis remodulates the host environment to promote virulence.

PubMed

Eddy, J L; Schroeder, J A; Zimbler, D L; Caulfield, A J; Lathem, W W

2016-09-01

Essentials Effect of plasminogen activator inhibitor (PAI)-1 on plague and its Y. pestis cleavage is unknown. An intranasal mouse model of infection was used to determine the role of PAI-1 in pneumonic plague. PAI-1 is cleaved and inactivated by the Pla protease of Y. pestis in the lung airspace. PAI-1 impacts both bacterial outgrowth and the immune response to respiratory Y. pestis infection. Click to hear Dr Bock discuss pathogen activators of plasminogen. Background The hemostatic regulator plasminogen activator inhibitor-1 (PAI-1) inactivates endogenous plasminogen activators and aids in the immune response to bacterial infection. Yersinia pestis, the causative agent of plague, produces the Pla protease, a virulence factor that is required during plague. However, the specific hemostatic proteins cleaved by Pla in vivo that contribute to pathogenesis have not yet been fully elucidated. Objectives To determine whether PAI-1 is cleaved by the Pla protease during pneumonic plague, and to define the impact of PAI-1 on Y. pestis respiratory infection in the presence or absence of Pla. Methods An intranasal mouse model of pneumonic plague was used to assess the levels of total and active PAI-1 in the lung airspace, and the impact of PAI-1 deficiency on bacterial pathogenesis, the host immune response and plasmin generation following infection with wild-type or ∆pla Y. pestis. Results We found that Y. pestis cleaves and inactivates PAI-1 in the lungs in a Pla-dependent manner. The loss of PAI-1 enhances Y. pestis outgrowth in the absence of Pla, and is associated with increased conversion of plasminogen to plasmin. Furthermore, we found that PAI-1 regulates immune cell recruitment, cytokine production and tissue permeability during pneumonic plague. Conclusions Our data demonstrate that PAI-1 is an in vivo target of the Pla protease in the lungs, and that PAI-1 is a key regulator of the pulmonary innate immune response. We conclude that the inactivation of PAI-1 by Y. pestis alters the host environment to promote virulence during pneumonic plague. © 2016 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

Corneal Expression of SLURP-1 by Age, Sex, Genetic Strain, and Ocular Surface Health

PubMed Central

Swamynathan, Sudha; Delp, Emili E.; Harvey, Stephen A. K.; Loughner, Chelsea L.; Raju, Leela; Swamynathan, Shivalingappa K.

2015-01-01

Purpose Although secreted Ly6/urokinase-type plasminogen activator receptor–related protein-1 (Slurp1) transcript is highly abundant in the mouse cornea, corresponding protein expression remains uncharacterized. Also, SLURP1 was undetected in previous tear proteomics studies, resulting in ambiguity about its baseline levels. Here, we examine mouse corneal Slurp1 expression in different sexes, age groups, strains, and health conditions, and quantify SLURP1 in human tears from healthy or inflamed ocular surfaces. Methods Expression of Slurp1 in embryonic day-13 (E13), E16, postnatal day-1 (PN1), PN10, PN20, and PN70 Balb/C, FVBN, C57Bl/6, and DBA/2J mouse corneas, Klf4Δ/ΔCE corneas with corneal epithelial–specific ablation of Klf4, migrating cells in wild-type corneal epithelial wound edge, and in corneas exposed to pathogen-associated molecular patterns (PAMPs) poly(I:C), zymosan-A, or Pam3Csk4 was examined by QPCR, immunoblots, and immunofluorescent staining. Human SLURP1 levels were quantified by ELISA in tears from 34 men and women aged 18 to 80 years. Results Expression of Slurp1, comparable in different strains and sexes, was low in E13, E16, PN1, and PN10 mouse corneas, and increased rapidly after eyelid opening in a Klf4-dependent manner. We found Slurp1 was downregulated in corneas exposed to PAMPs, and in migrating cells at the wound edge. Human SLURP1 expression, comparable in different sexes and age groups, was significantly decreased in tears from inflamed ocular surfaces (0.34%) than those from healthy individuals (0.77%). Conclusions These data describe the influence of age, sex, genetic background, and ocular surface health on mouse corneal expression of Slurp1, establish the baseline for human tear SLURP1 expression, and identify SLURP1 as a useful diagnostic and/or therapeutic target for inflammatory ocular surface disorders. PMID:26670825

Plasminogen replacement therapy for the treatment of children and adults with congenital plasminogen deficiency

PubMed Central

Nakar, Charles; Parker, Joseph M.; Albert, Gary R.; Moran, John E.; Thibaudeau, Karen; Thukral, Neelam; Hardesty, Brandon M.; Laurin, Pierre; Sandset, Per Morten

2018-01-01

Congenital plasminogen deficiency is caused by mutations in PLG, the gene coding for production of the zymogen plasminogen, and is an ultrarare disorder associated with abnormal accumulation or growth of fibrin-rich pseudomembranous lesions on mucous membranes. Left untreated, these lesions may impair organ function and impact quality of life. Plasminogen replacement therapy should provide an effective treatment of the manifestations of congenital plasminogen deficiency. An open-label phase 2/3 study of human Glu-plasminogen administered IV at 6.6 mg/kg every 2 to 4 days in 15 patients with congenital plasminogen deficiency is ongoing. Reported here are data on 14 patients who completed at least 12 weeks of treatment. The primary end point was an increase in trough plasminogen activity levels by at least an absolute 10% above baseline. The secondary end point was clinical success, defined as ≥50% improvement in lesion number/size or functionality impact from baseline. All patients achieved at least an absolute 10% increase in trough plasminogen activity above baseline. Clinical success was observed in all patients with clinically visible (conjunctiva and gingiva), nonvisible (nasopharynx, bronchus, colon, kidney, cervix, and vagina), and wound-healing manifestations of the disease. Therapeutic effects were rapid, as all but 2 lesions resolved or improved after 4 weeks of treatment. Human Glu-plasminogen was well tolerated in both children and adults. This study provides critical first evidence of the clinical utility of ongoing replacement therapy with human Glu-plasminogen for the treatment of children and adults with congenital plasminogen deficiency. This trial was registered at www.clinicaltrials.gov as #NCT02690714. PMID:29321155

Heat shock proteins HSP70 and MRJ cooperatively regulate cell adhesion and migration through urokinase receptor.

PubMed

Lin, Yuli; Peng, Nana; Zhuang, Hongqin; Zhang, Di; Wang, Yao; Hua, Zi-Chun

2014-08-30

The urokinase-type plasminogen activator receptor (uPAR) is an important regulator of ECM proteolysis, cell-ECM interactions and cell signaling. uPAR and heat shock proteins HSP70 and MRJ (DNAJB6) have been implicated in tumor growth and metastasis. We have reported recently that MRJ (DNAJB6, a heat shock protein) can interact with uPAR and enhance cell adhesion. Here, we identified another heat shock protein HSP70 as a novel uPAR-interacting protein. We performed co-immunoprecipitation in human embryonic kidney (HEK) 293 and colon cancer HCT116 cells as well as immunofluorence assays in HEK293 cells stably transfected with uPAR to investigate the association of suPAR with HSP70/MRJ. To understand the biological functions of the triple complex of suPAR/HSP70/MRJ, we determined whether HSP70 and/or MRJ regulated uPAR-mediated cell invasion, migration, adhesion to vitronectin and MAPK pathway in two pair of human tumor cells (uPAR negative HEK293 cells vs HEK293 cells stably transfected with uPAR and HCT116 cells stably transfected with antisense-uPAR vs HCT116 mock cells transfected with vector only) using transwell assay, wound healing assay, quantitative RT-PCR analyzing mmp2 and mmp9 transcription levels, cell adhesion assay and Western blotting assay. HSP70 and MRJ formed a triple complex with uPAR and over-expression of MRJ enhanced the interaction between HSP70 and uPAR, while knockdown of MRJ decreased soluble uPAR in HCT116 cells (P < 0.05) and reduced the formation of the triple complex, suggesting that MRJ may act as an uPAR-specific adaptor protein to link uPAR to HSP70. Further experiments showed that knockdown of HSP70 and/or MRJ by siRNA inhibited uPAR-mediated cell adhesion to vitronectin as well as suppressed cell invasion and migration. Knockdown of HSP70 and/or MRJ inhibited expression of invasion related genes mmp2 and mmp9. Finally, HSP70 and/or MRJ up-regulated phosphorylation levels of ERK1/2 and FAK suggesting MAPK pathway was involved. All the biological function experiments in cell level showed an additive effect when HSP70 and MRJ were regulated simultaneously indicating their collaborated regulation effects on uPAR. These findings may offer a novel insight into the interactions between uPAR and HSP70/MRJ and their functions in cell adhesion and migration may provide more understanding of the roles in regulating cancer metastasis.

BreastDefend™ prevents breast-to-lung cancer metastases in an orthotopic animal model of triple-negative human breast cancer

PubMed Central

JIANG, JIAHUA; THYAGARAJAN-SAHU, ANITA; LOGANATHAN, JAGADISH; ELIAZ, ISAAC; TERRY, COLIN; SANDUSKY, GEORGE E.; SLIVA, DANIEL

2012-01-01

We have recently demonstrated that a natural dietary supplement BreastDefend (BD), which contains extracts from medicinal mushrooms (Coriolus versicolor, Ganoderma lucidum, Phellinus linteus), medicinal herbs (Scutellaria barbata, Astragalus membranaceus, Curcuma longa), and purified biologically active nutritional compounds (diindolylmethane and quercetin), inhibits proliferation and metastatic behavior of MDA-MB-231 invasive human breast cancer cells in vitro. In the present study, we evaluated whether BD suppresses growth and breast-to lung cancer metastasis in an orthotopic model of human breast cancer cells implanted in mice. Oral application of BD (100 mg/kg of body weight for 4 weeks) by intragastric gavage did not affect body weight or activity of liver enzymes and did not show any sign of toxicity in liver, spleen, kidney, lung and heart tissues in mice. Moreover, BD significantly decreased the change in tumor volume over time compared to the control group (p=0.002). BD treatment also markedly decreased the incidence of breast-to-lung cancer metastasis from 67% (control) to 20% (BD) (p<0.05) and the number of metastases from 2.8 (0.0, 48.0) in the control group to 0.0 (0.0, 14.2) in the BD treatment group (p<0.05). Finally, anti-metastatic activity of BD in vivo was further confirmed by the downregulation of expression of PLAU (urokinase plasminogen activator, uPA) and CXCR4 (C-X-C chemokine receptor-4) genes in breast tumors. In conclusion, BD may be considered as a biological therapeutic agent against invasive breast cancers. PMID:22842551

Dysfunction of annexin A2 contributes to hyperglycaemia-induced loss of human endothelial cell surface fibrinolytic activity.

PubMed

Dai, Haibin; Yu, Zhanyang; Fan, Xiang; Liu, Ning; Yan, Min; Chen, Zhong; Lo, Eng H; Hajjar, Katherine A; Wang, Xiaoying

2013-06-01

Hyperglycaemia impairs fibrinolytic activity on the surface of endothelial cells, but the underlying mechanisms are not fully understood. In this study, we tested the hypothesis that hyperglycaemia causes dysfunction of the endothelial membrane protein annexin A2, thereby leading to an overall reduction of fibrinolytic activity. Hyperglycaemia for 7 days significantly reduced cell surface fibrinolytic activity in human brain microvascular endothelial cells (HBMEC). Hyperglycaemia also decreased tissue type plasminogen activator (t-PA), plasminogen, and annexin A2 mRNA and protein expression, while increasing plasminogen activator inhibitor-1 (PAI-1). No changes in p11 mRNA or protein expression were detected. Hyperglycaemia significantly increased AGE-modified forms of total cellular and membrane annexin A2. The hyperglycemia-associated reduction in fibrinolytic activity was fully restored upon incubation with recombinant annexin A2 (rA2), but not AGE-modified annexin A2 or exogenous t-PA. Hyperglycaemia decreased t-PA, upregulated PAI-1 and induced AGE-related disruption of annexin A2 function, all of which contributed to the overall reduction in endothelial cell surface fibrinolytic activity. Further investigations to elucidate the underlying molecular mechanisms and pathophysiological implications of A2 derivatisation might ultimately lead to a better understanding of mechanisms of impaired vascular fibrinolysis, and to development of new interventional strategies for the thrombotic vascular complications in diabetes.

Dysfunction of annexin A2 contributes to hyperglycaemia-induced loss of human endothelial cell surface fibrinolytic activity

PubMed Central

Dai, Haibin; Yu, Zhanyang; Fan, Xiang; Liu, Ning; Yan, Min; Chen, Zhong; Lo, Eng H.; Hajjar, Katherine A.; Wang, Xiaoying

2014-01-01

Summary Hyperglycaemia impairs fibrinolytic activity on the surface of endothelial cells, but the underlying mechanisms are not fully understood. In this study, we tested the hypothesis that hyperglycaemia causes dysfunction of the endothelial membrane protein annexin A2, thereby leading to an overall reduction of fibrinolytic activity. Hyperglycaemia for 7 days significantly reduced cell surface fibrinolytic activity in human brain microvascular endothelial cells (HBMEC). Hyperglycaemia also decreased tissue type plasminogen activator (t-PA), plasminogen, and annexin A2 mRNA and protein expression, while increasing plasminogen activator inhibitor-1 (PAI-1). No changes in p11 mRNA or protein expression were detected. Hyperglycaemia significantly increased AGE-modified forms of total cellular and membrane annexin A2. The hyperglycemia-associated reduction in fibrinolytic activity was fully restored upon incubation with recombinant annexin A2 (rA2), but not AGE-modified annexin A2 or exogenous t-PA. Hyperglycaemia decreased t-PA, upregulated PAI-1 and induced AGE-related disruption of annexin A2 function, all of which contributed to the overall reduction in endothelial cell surface fibrinolytic activity. Further investigations to elucidate the underlying molecular mechanisms and pathophysiological implications of A2 derivatisation might ultimately lead to a better understanding of mechanisms of impaired vascular fibrinolysis, and to development of new interventional strategies for the thrombotic vascular complications in diabetes. PMID:23572070

The story of an exceptional serine protease, tissue-type plasminogen activator (tPA).

PubMed

Hébert, M; Lesept, F; Vivien, D; Macrez, R

2016-03-01

The only acute treatment of ischemic stroke approved by the health authorities is tissue recombinant plasminogen activator (tPA)-induced thrombolysis. Under physiological conditions, tPA, belonging to the serine protease family, is secreted by endothelial and brain cells (neurons, astrocytes, microglia, oligodendrocytes). Although revascularisation induced by tPA is beneficial during a stroke, research over the past 20 years shows that tPA can also be deleterious for the brain parenchyma. Thus, in this review of the literature, after a brief history on the discovery of tPA, we reviewed current knowledge of mechanisms by which tPA can influence brain function in physiological and pathological conditions. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Quantitative Method of Measuring Metastatic Activity

NASA Technical Reports Server (NTRS)

Morrison, Dennis R. (Inventor)

1999-01-01

The metastatic potential of tumors can be evaluated by the quantitative detection of urokinase and DNA. The cell sample selected for examination is analyzed for the presence of high levels of urokinase and abnormal DNA using analytical flow cytometry and digital image analysis. Other factors such as membrane associated uroldnase, increased DNA synthesis rates and certain receptors can be used in the method for detection of potentially invasive tumors.

Immunoregulatory Protein Profiles of Necrotizing Enterocolitis versus Spontaneous Intestinal Perforation in Preterm Infants

PubMed Central

Leung, Fiona Wan Lun; Lam, Hugh Simon; Tam, Yuk Him; To, Ka Fai; Cheung, Hon Ming; Leung, Kam Tong; Poon, Terence Chuen Wai; Lee, Kim Hung; Li, Karen; Fok, Tai Fai

2012-01-01

Necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) are the most common acute surgical emergencies associated with high morbidity and mortality in preterm infants. We aimed to compare the profiles of immunoregulatory proteins and identify novel mediators in plasma of NEC and SIP infants. We also investigated the expression of target genes in resected intestinal tissues and an enterocyte cell line. Using Cytokine Antibody Array assay, we reported the first comparative profiles of immunoregulatory proteins in plasma of NEC and SIP infants, and showed that dysregulated proteins belonged to functionally diversified categories, including pro- and anti-inflammation, angiogenesis, cell growth, wound healing, anti-apoptosis, cell adhesion and extracellular matrix reorganization. Validation by ELISA confirmed significantly higher concentrations of interleukin (IL)-6, angiopoietin (Ang)-2, soluble type II interleukin-1 receptor (sIL-1RII), and soluble urokinase-type plasminogen activator receptor (suPAR) in NEC infants compared with gestational age-matched control, and a lower level of an epidermal growth factor receptor, secreted form of receptor tyrosine-protein kinase ErbB3 (sErbB3), compared with SIP infants. mRNA expressions of IL1-RII and uPAR were up-regulated in resected bowel tissues from NEC infants, indicating that immunoregulation also occurred at the cellular level. In FHs-74 Int cells, Ang-2, IL1-RII and uPAR mRNA expressions were significantly induced by the combined treatment with lipopolysaccharide (LPS) and platelet activating factor (PAF). Our study provided plasmatic signatures of immunoregulatory proteins in NEC and SIP infants, and demonstrated involvement of multiple functional pathways. The magnitude of changes in these proteins was significantly more extensive in NEC infants, reflecting the different nature of injury and/or severity of inflammation. We speculate that dysregulation of IL-6, Ang-2, IL-1RII and uPAR occurred at both systemic and cellular levels, and probably mediated via LPS and endogeneous PAF signals. Such exaggerated immunologic responses may account for the high morbidity and mortality in NEC compared with SIP patients. PMID:22606320

The soluble Decoy Receptor 3 is regulated by a PI3K-dependent mechanism and promotes migration and invasion in renal cell carcinoma.

PubMed

Weissinger, Daniel; Tagscherer, Katrin E; Macher-Göppinger, Stephan; Haferkamp, Axel; Wagener, Nina; Roth, Wilfried

2013-10-10

Overexpression of Decoy Receptor 3 (DcR3), a soluble member of the tumor necrosis factor receptor superfamily, is a common event in several types of cancer. In renal cell carcinoma (RCC), DcR3 overexpression is associated with lymph node and distant metastasis as well as a poor prognosis. However, the functional role and regulation of DcR3 expression in RCC is so far unknown. Modulation of DcR3 expression by siRNA and ectopic gene expression, respectively, was performed in ACHN and 769-P RCC cell lines. Functional effects of a modulated DcR3 expression were analyzed with regard to migration, invasion, adhesion, clonogenicity, and proliferation. Furthermore, quantitative RT-PCR and immunoblot analyses were performed to evaluate the expression of downstream mediators of DcR3. In further experiments, luciferase assays, quantitative RT-PCR and immunoblot analyses were applied to study the regulation of DcR3 expression in RCC. Additionally, an ex vivo tissue slice culture technique combined with immunohistochemistry was used to study the regulation of DcR3 expression in human RCC specimens. Here, we show that DcR3 promotes adhesion, migration and invasiveness of RCC cells. The DcR3-dependent increase in cellular invasiveness is accompanied with an up-regulation of integrin alpha 4, matrixmetalloproteinase 7 and urokinase plasminogen activator (uPA). Further, we identified a signaling pathway regulating DcR3 expression in RCC. Using in vitro experiments as well as an ex vivo RCC tissue slice culture model, we demonstrate that expression of DcR3 is regulated in a PI3K/AKT-dependent manner involving the transcription factor nuclear factor of activated T-cells (NFAT). Taken together, our results identify DcR3 as a key driver of tumor cell dissemination and suggest DcR3 as a promising target for rational therapy of RCC.

Helodermatine, a kallikrein-like, hypotensive enzyme from the venom of Heloderma horridum horridum (Mexican beaded lizard)

PubMed Central

1986-01-01

We have purified and characterized the major N-benzoyl-L-arginine ethyl ester hydrolase from the venom of Heloderma horridum horridum. The enzyme belongs to the serine proteinase family, and its activity vs. peptide amide substrates and human high-molecular-weight kininogen suggests a similarity to the family of kallikreins. This interpretation is corroborated by its reactivity with the natural inhibitors soybean trypsin inhibitor and Kunitz-type bovine pancreatic trypsin inhibitor (aprotinin). Injection of the enzyme (2-16 micrograms/kg) into anesthetized rabbits leads to a rapid dose-dependent transient decrease of the arterial blood pressure. Like glandular kallikrein it specifically converts single-chain tissue type plasminogen activator into its double chain form. In contrast to other kallikrein-like enzymes from snake venoms it shows no thrombin-like or plasminogen activator activity. The enzyme is a single-chain glycoprotein (Mr 63,000). The N-terminal sequence revealed significant homology to pig pancreatic kallikrein and to kallikrein like enzymes from Crotalus atrox and Crotalus adamanteus venom. This enzyme, which we name Helodermatine, is the first purified from Sauria with kallikrein-like properties. PMID:3537191

Plasminogen Activator Production Accompanies Loss of Anchorage Regulation in Transformation of Primary Rat Embryo Cells by Simian Virus 40

PubMed Central

Pollack, R.; Risser, R.; Conlon, S.; Rifkin, D.

1974-01-01

We have isolated several lines of rat embryo cells transformed by simian virus 40. All these lines are fully transformed with regard to saturation density and serum sensitivity, but they differ greatly in their anchorage dependence, as assayed by efficiency of plating in methyl cellulose suspension. This set of lines reveals a consistent relation of plasminogen activator production to plating efficiency in methyl cellulose. T-antigen-positive transformed lines that synthesize activator grow in methyl cellulose suspension, while T-antigen-positive transformed lines that do not synthesize activator fail to form colonies in suspension. Normal rat embryo cells produce very little plasminogen activator and do not grow in methyl cellulose. Sera that permit high levels of plasmin formation and activity support growth in semi-solid medium better than sera whose plasminogen is activated poorly and/or sera that contain inhibitors to plasmin. PMID:4373730

Plasminogen activator inhibitor type 1 gene is located at region q21. 3-q22 of chromosome 7 and genetically linked with cystic fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klinger, K.W.; Winqvist, R.; Riccio, A.

1987-12-01

The regional chromosomal location of the human gene for plasminogen activator inhibitor type 1 (PAI1) was determined by three independent methods of gene mapping. PAI1 was localized first to 7cen-q32 and then to 7q21.3-q22 by Southern blot hybridization analysis of a panel of human and mouse somatic cell hybrids with a PAI1 cDNA probe and in situ hybridization, respectively. The authors frequent HindIII restriction fragment length polymorphism (RFLP) of the PAI1 gene with an information content of 0.369. In family studies using this polymorphism, genetic linkage was found between PAI1 and the loci for erythropoietin (EPO), paraoxonase (PON), the metmore » protooncogene (MET), and cystic fibrosis (CF), all previously assigned to the middle part of the long arm of chromosome 7. The linkage with EPO was closest with an estimated genetic distance of 3 centimorgans, whereas that to CF was 20 centimorgans. A three-point genetic linkage analysis and data from previous studies showed that the most likely order of these loci is EPO, PAI1, PON, (MET, CF), with PAI1 being located centromeric to CF. The PAI1 RFLP may prove to be valuable in ordering genetic markers in the CF-linkage group and may also be valuable in genetic analysis of plasminogen activation-related diseases, such as certain thromboembolic disorders and cancer.« less

Biobusiness in the pharmaceutical industry.

PubMed

Werner, R G

1987-09-01

Although conventional biotechnology used for the synthesis of antibiotics, vitamins, amino acids, nucleotides, enzyme inhibitors and immunomodulating compounds has still a major impact in the production of pharmaceutical compounds, the importance of the new biotechnology is increasing. Whereas in conventional biotechnology naturally occurring strains are screened for production of pharmacologically active compounds, in new biotechnology known organisms are programmed by genetic engineering to produce a distinct protein or glycoprotein of human origin for substitution therapy. Such complex compounds from new biotechnology can be divided into products which might replace compounds which are already on the market by safer recombinant products such as human insulin, human growth hormone, urokinase, factor VIII and products which are new on the market such as interferons, lymphokines, tissue plasminogen activator, oligonucleotide probes, monoclonal antibodies and subunit vaccines. However, only a few of these recombinant products have reached the market such as human insulin, interferon alpha, interferon beta, human growth hormone and recombivax HB. In most cases, depending on the difficulties in demonstrating clinical efficacy, the investigated drugs have reached the marketing phase much faster than conventional chemical drugs. Return on investment of biotechnical produced pharmaceutics mainly depends on the issues of whether the product has to compete with chemically synthesized drugs, whether it is totally new but competes with other bioproducts, whether it is exceptional but the proof of clinical efficacy is difficult, or whether it is totally new and clinical studies are promising.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of markers of bacterial translocation with immune activation in decompensated cirrhosis.

PubMed

Mortensen, Christian; Jensen, Jørgen Skov; Hobolth, Lise; Dam-Larsen, Sanne; Madsen, Bjørn S; Andersen, Ove; Møller, Søren; Bendtsen, Flemming

2014-12-01

Bacterial translocation (BT) may cause infections, in particular, spontaneous bacterial peritonitis (SBP). In the absence of overt infection, BT may further stimulate the immune system and contribute to haemodynamic alterations and complications. Bacterial DNA (bDNA) is claimed to be a promising surrogate marker for BT, although its clinical relevance has been questioned. In 38 cirrhotic patients with and without SBP, bDNA in blood and ascites were assessed by 16S rDNA quantitative PCR. Levels of lipopolysaccharide-binding protein in plasma and highly sensitive C-reactive protein, tumour necrosis factor-α, soluble urokinase plasminogen activating receptor, interleukin-6, interleukin 8, interferon-γ inducible protein-10 and vascular endothelial growth factor in plasma and ascites were measured by multiplex cytokine and ELISA assays. In patients without signs of SBP or positive cultures, we found a high frequency of bDNA but low concordance of bDNA between blood and ascites. Markers of inflammation were not significantly different between blood bDNA-positive (22%), ascites bDNA-positive (52%), and bDNA-negative patients. The 16S rDNA PCR failed to show bDNA in two out of six samples with SBP. Sequencing of positive samples did not determine the source of bDNA. bDNA as assessed by this PCR method was largely unrelated to markers of inflammation and does not seem to be of clinical value in the diagnosis of SBP. According to our results, bDNA is not a reliable marker of BT.

Expression and activity analysis of a new fusion protein targeting ovarian cancer cells.

PubMed

Su, Manman; Chang, Weiqin; Wang, Dingding; Cui, Manhua; Lin, Yang; Wu, Shuying; Xu, Tianmin

2015-09-01

The aim of the present study was to develop a new therapeutic drug to improve the prognosis of ovarian cancer patients. Human urokinase-type plasminogen activator (uPA)17-34-kunitz-type protease inhibitor (KPI) eukaryotic expression vector was constructed and recombinant human uPA17-34-KPI (rhuPA17-34-KPI) in P. pastoris was expressed. In the present study, the DNA sequences that encode uPA 17-34 amino acids were created according to the native amino acids sequence and inserted into the KPI-pPICZαC vector, which was constructed. Then, uPA17‑34-KPI-pPICZαC was transformed into P. pastoris X-33, and rhuPA17-34-KPI was expressed by induction of methanol. The bioactivities of a recombinant fusion protein were detected with trypsin inhibition analysis, and the inhibitory effects on the growth of ovarian cancer cells were identified using the TUNEL assay, in vitro wound‑healing assay and Matrigel model analysis. The results of the DNA sequence analysis of the recombinant vector uPA17-34-KPI‑pPICZα demonstrated that the DNA‑encoding human uPA 17-34 amino acids, 285-288 amino acids of amyloid precursor protein (APP) and 1-57 amino acids of KPI were correctly inserted into the pPICZαC vector. Following induction by methonal, the fusion protein with a molecular weight of 8.8 kDa was observed using SDS-PAGE and western blot analysis. RhuPA17-34-KPI was expressed in P. pastoris with a yield of 50 mg/l in a 50-ml tube. The recombinant fusion protein was able to inhibit the activity of trypsin, inhibit growth and induce apoptosis of SKOV3 cells, and inhibit the invasion and metastasis of ovarian cancer cells. By considering uPA17-34 amino acid specific binding uPAR as the targeted part of fusion protein and utilizing the serine protease inhibitor activity of KPI, it was found that the recombinant fusion protein uPA17-34-KPI inhibited the invasion and metastasis of ovarian tumors, and may therefore be regarded as effective in targeted treatment.

Studies on a complex mechanism for the activation of plasminogen by kaolin and by chloroform: the participation of Hageman factor and additional cofactors

PubMed Central

Ogston, Derek; Ogston, C. Marie; Ratnoff, Oscar D.; Forbes, Charles D.

1969-01-01

As demonstrated by others, fibrinolytic activity was generated in diluted, acidified normal plasma exposed to kaolin, a process requiring Hageman factor (Factor XII). Generation was impaired by adsorbing plasma with glass or similar agents under conditions which did not deplete its content of Hageman factor or plasminogen. The defect could be repaired by addition of a noneuglobulin fraction of plasma or an agent or agents eluted from diatomaceous earth which had been exposed to normal plasma. The restorative agent, tentatively called Hageman factor-cofactor, was partially purified by chromatography and had an apparent molecular weight of approximately 165,000. It could be distinguished from plasma thromboplastin antecedent (Factor XI) and plasma kallikrein, other substrates of Hageman factor, and from the streptokinase-activated pro-activator of plasminogen. Evidence is presented that an additional component may be needed for the generation of fibrinolytic activity in mixtures containing Hageman factor, HF-cofactor, and plasminogen. The long-recognized generation of plasmin activity in chloroform-treated euglobulin fractions of plasma was found to be dependent upon the presence of Hageman factor. Whether chloroform activation of plasminogen requires Hageman factor-cofactor was not determined, but glass-adsorbed plasma, containing Hageman factor and plasminogen, did not generate appreciable fibrinolytic or caseinolytic activity. These studies emphasize the complex nature of the mechanisms which lead to the generation of plasmin in human plasma. PMID:4241814

Fibrinolytic agents in the management of posthemorrhagic hydrocephalus in preterm infants: the evidence.

PubMed

Haines, S J; Lapointe, M

1999-05-01

The objective of this study was to review current literature on the management of posthemorrhagic hydrocephalus in preterm infants with intraventricular administration of fibrinolytic agents; to this end a literature search was carried out electronically. The keywords used were "intraventricular hemorrhage" or "posthemorrhagic hydrocephalus" in combination with "fibrinolytic agent," "urokinase," "streptokinase," or "recombinant tissue plasminogen activator" and "intraventricular administration"; the search covered the years 1966-1998 and was restricted to English language papers and human subjects. It was supplemented by a search through the reference lists of the articles identified. Articles dealing with intracerebral hemorrhage or hematoma, intraventricular hemorrhage in adults, nontherapeutic issues and laboratory research were excluded. The articles included are summarized in evidence and evaluation tables. Five scientific publications evaluating the use of a fibrinolytic agent to manage posthemorrhagic hydrocephalus were retrieved. In the studies described in these reports, a total of 62 neonates received streptokinase, urokinase or r-tPA intraventricularly. No two of the regimens were identical in the drug used, method of administration and duration of therapy. The time before therapy was started ranged from 2 to 35 days after the ictus. Among the case series reported, three were small series with a total of 38 neonates. One other case series of 18 neonates compared the treatment group with an historical control group. All case series showed that endoventricular fibrinolytic therapy was practical. The proportion of cases in which shunt placement was performed ranged from 11% to 100%. Only one small prospective, randomized, controlled study was identified. That study was too small to allow useful conclusions. Overall, 3 cases of secondary intraventricular hemorrhage were reported. However, it was not possible to determine with certainty whether these episodes were related to the drug therapy itself. The reports suffer from inadequate study design, lack of descriptive information and short follow-up period. There is insufficient evidence to justify the claim that fibrinolytic agents administered intraventricularly in posthemorrhagic hydrocephalus are safe and effective. More evidence is needed to prove or disprove the effectiveness and safety of this form of therapy.

Attachment, invasion, chemotaxis, and proteinase expression of B16-BL6 melanoma cells exhibiting a low metastatic phenotype after exposure to dietary restriction of tyrosine and phenylalanine.

PubMed

Uhlenkott, C E; Huijzer, J C; Cardeiro, D J; Elstad, C A; Meadows, G G

1996-03-01

We previously reported that low levels of tyrosine (Tyr) and phenylalanine (Phe) alter the metastatic phenotype of B16-BL6 (BL6) murine melanoma and select for tumor cell populations with decreased lung colonizing ability. To more specifically characterize the effects of Tyr and Phe restriction on the malignant phenotype of BL6, we investigated in vitro attachment, invasion, proteinase expression, and chemotaxis of high and low metastatic BL6 variants. High metastatic variant cells were isolated from subcutaneous tumors of mice fed a nutritionally complete diet (ND cells) and low metastatic variant cells were isolated from mice fed a diet restricted in Tyr and Phe (LTP cells). Results indicate that attachment to reconstituted basement membrane (Matrigel) was significantly reduced in LTP cells as compared to ND cells. Attachment to collagen IV, laminin, and fibronectin were similar between the two variants. Invasion through Matrigel and growth factor-reduced Matrigel were significantly decreased in LTP cells as compared to ND cells. Zymography revealed the presence of M(r) 92,000 and M(r) 72,000 progelatinases, tissue plasminogen activator, and urokinase plasminogen activator in the conditioned medium of both variants; however, there were no differences in activity of these secreted proteinases between the two variants. Growth of the variants on growth factor-reduced Matrigel similarly induced expression of the M(r) 92,000 progelatinase. The variants exhibited similar chemotactic responses toward laminin. However, the chemotactic response toward fibronectin by LTP cells was significantly increased. MFR5, a monoclonal antibody which selectively blocks function of the alpha 5 chain of the alpha 5 beta 1 integrin, VLA-5, decreased the chemotactic response toward fibronectin of ND cells by 37%; the chemotactic response by LTP cells was reduced by 49%. This effect was specific for fibronectin-mediated chemotaxis since the chemotaxis toward laminin and invasion through Matrigel were not altered by the presence of MFR5. The surface expression of VLA-5 was significantly increased in LTP cells as compared to ND cells by flow cytometric analysis. These observations suggest that limitation of Tyr and Phe either directly modifies BL6 or selects for subpopulations with altered in vitro invasion, chemotaxis, and integrin expression.

Photonic Activation of Plasminogen Induced by Low Dose UVB

PubMed Central

Correia, Manuel; Snabe, Torben; Thiagarajan, Viruthachalam; Petersen, Steffen Bjørn; Campos, Sara R. R.; Baptista, António M.; Neves-Petersen, Maria Teresa

2015-01-01

Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity was observed after 10 min illumination of human plasminogen. Irradiance levels used are in the same order of magnitude of the UVB solar irradiance. Activation is correlated with light induced disruption of disulphide bridges upon UVB excitation of the aromatic residues and with the formation of photochemical products, e.g. dityrosine and N-formylkynurenine. Most of the protein fold is maintained after 10 min illumination since no major changes are observed in the near-UV CD spectrum. Far-UV CD shows loss of secondary structure after illumination (33.4% signal loss at 206 nm). Thermal unfolding CD studies show that plasminogen retains a native like cooperative transition at ~70 ºC after UV-illumination. We propose that UVB activation of plasminogen occurs upon photo-cleavage of a functional allosteric disulphide bond, Cys737-Cys765, located in the catalytic domain and in van der Waals contact with Trp761 (4.3 Å). Such proximity makes its disruption very likely, which may occur upon electron transfer from excited Trp761. Reduction of Cys737-Cys765 will result in likely conformational changes in the catalytic site. Molecular dynamics simulations reveal that reduction of Cys737-Cys765 in plasminogen leads to an increase of the fluctuations of loop 760–765, the S1-entrance frame located close to the active site. These fluctuations affect the range of solvent exposure of the catalytic triad, particularly of Asp646 and Ser74, which acquire an exposure profile similar to the values in plasmin. The presented photonic mechanism of plasminogen activation has the potential to be used in clinical applications, possibly together with other enzymatic treatments for the elimination of blood clots. PMID:25635856

Ethnic Comparison of Clinical Characteristics and Ischemic Stroke Subtypes Among Young Adult Patients With Stroke in Hawaii.

PubMed

Nakagawa, Kazuma; Ito, Cherisse S; King, Sage L

2017-01-01

Native Hawaiians and other Pacific Islanders (NHOPI) with ischemic stroke have younger age of stroke onset compared with whites. However, ethnic differences in stroke subtypes in this population have been inadequately studied. Consecutive young adult patients (aged ≤55 years) who were hospitalized for ischemic stroke between 2006 and 2012 at a tertiary center in Honolulu were studied. Clinical characteristics and stroke subtypes based on pathophysiological TOAST classification (Trial of Org 10172) of NHOPI and Asians were compared with whites. A total of 427 consecutive young adult (mean age, 46.7±7.8 years) patients (NHOPI 45%, Asians 38%, and whites 17%) were studied. NHOPI had a higher prevalence of hypertension, diabetes mellitus, prosthetic valve, higher body mass index, hemoglobin A1c, and lower high-density lipoprotein than whites (all P<0.05). Stroke subtype distribution was not different between the ethnic groups. Specifically, the prevalence of small-vessel disease was similar between NHOPI (26.6%), whites (28.4%), and Asians (24.8%). In the univariate analyses, the use of intravenous tissue-type plasminogen activator was lower among NHOPI (4.7%; P=0.01) and Asians (3.1%; P=0.002) than among whites (12.5%). In the multivariable model, NHOPI (odds ratio, 0.35; 95% confidence interval, 0.12-0.98) and Asians (odds ratio, 0.23; 95% confidence interval, 0.07-0.74) were less likely to be treated with intravenous tissue-type plasminogen activator than whites. NHOPI have greater cardiovascular risk factors than whites, but there were no differences in stroke subtypes between the ethnic groups. Furthermore, NHOPI and Asians may be less likely to be treated with intravenous tissue-type plasminogen activator than whites. © 2016 American Heart Association, Inc.

Improving door-to-needle times: a single center validation of the target stroke hypothesis.

PubMed

Ruff, Ilana M; Ali, Syed F; Goldstein, Joshua N; Lev, Michael; Copen, William A; McIntyre, Joyce; Rost, Natalia S; Schwamm, Lee H

2014-02-01

National guidelines recommend imaging within 25 minutes of emergency department arrival and intravenous tissue-type plasminogen activator within 60 minutes of emergency department arrival for patients with acute stroke. In 2007, we implemented a new institutional acute stroke care model to include 10 best practices and evaluated the effect of this intervention on improving door-to-computed tomography (CT) and door-to-needle (DTN) times at our hospital. We compared patients who presented directly to our hospital with acute ischemic stroke in the preintervention (2003-2006) and postintervention (2008-2011) periods. We did not include 2007, the year that the new protocol was established. Predictors of DTN ≤60 minutes before and after the intervention were assessed using χ(2) for categorical variables, and t test and Wilcoxon signed-rank test for continuous variables. Among 2595 patients with acute stroke, 284 (11%) received intravenous tissue-type plasminogen activator. For patients arriving within an intravenous tissue-type plasminogen activator window, door-to-CT <25 improved from 26.7% pre intervention to 52.3% post intervention (P<0.001). Similarly, the percentage of patients with DTN <60 doubled from 32.4% to 70.3% (P<0.001). Patients with DTN ≤60 did not differ significantly with respect to demographics, comorbidities, or National Institutes of Health Stroke Scale score in comparison with those treated after 60 minutes. Door-to-CT and DTN times improved dramatically after applying 10 best practices, all of which were later incorporated into the Target Stroke Guidelines created by the American Heart Association. The only factor that significantly affected DTN60 was the intervention itself, indicating that these best practices can result in improved DTN times.

Antiplatelet, anticoagulant, and profibrinolytic activities of withaferin A.

PubMed

Ku, Sae-Kwang; Bae, Jong-Sup

2014-03-01

Withaferin A (WFA), an active compound from Withania somnifera, is widely researched for its anti-inflammatory, cardioactive and central nervous system effects. However, antiplatelet, anticoagulant, and profibrinolytic properties of WFA have not been studied. In this study, the anticoagulant activities of WFA were measured by monitoring activated partial thromboplastin-time (aPTT), prothrombin time (PT), fibrin polymerization, platelet aggregation, thrombus formation, and the activities of cell-based thrombin and activated factor X (FXa). The effects of WFA on the expressions of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were also tested in tumor necrosis factor-α (TNF-α) activated human umbilical vein endothelial cells (HUVECs). Our data showed that WFA inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation, FeCl3-induced thrombus formation, prolonged aPTT and PT significantly and inhibited the activities and production of thrombin and FXa. WFA prolonged in vivo and ex vivo bleeding time and inhibited TNF-α induced PAI-1 production. Furthermore, PAI-1/t-PA ratio was significantly decreased by WFA. Collectively, these results indicate that WFA possesses antithrombotic activities and suggest that the current study could provide bases for the development of new anticoagulant agents. Copyright © 2014 Elsevier Inc. All rights reserved.

A Reexamination of Active and Passive Tumor Targeting by Using Rod-Shaped Gold Nanocrystals and Covalently Conjugated Peptide Ligands

PubMed Central

Huang, Xiaohua; Peng, Xianghong; Wang, Yiqing; Wang, Yuxiang; Shin, Dong M.; El-Sayed, Mostafa A.; Nie, Shuming

2010-01-01

The targeted delivery of nanoparticles to solid tumors is one of the most important and challenging problems in cancer nanomedicine, but the detailed delivery mechanisms and design principles are still not well understood. Here we report quantitative tumor uptake studies for a class of elongated gold nanocrystals (called nanorods) that are covalently conjugated to tumor-targeting peptides. A major advantage in using gold as a “tracer” is that the accumulated gold in tumors and other organs can be quantitatively determined by elemental mass spectrometry (gold is not a natural element found in animals). Thus, colloidal gold nanorods are stabilized with a layer of polyethylene glycols (PEGs), and are conjugated to three different ligands: (i) a single-chain variable fragment (ScFv) peptide that recognizes the epidermal growth factor receptor (EGFR); (ii) an amino terminal fragment (ATF) peptide that recognizes the urokinase plasminogen activator receptor (uPAR); and (iii) a cyclic RGD peptide that recognizes the avb3 integrin receptor. Quantitative pharmacokinetic and biodistribution data show that these targeting ligands only marginally improve the total gold accumulation in xenograft tumor models in comparison with nontargeted controls, but their use could greatly alter the intracellular and extracellular nanoparticle distributions. When the gold nanorods are administered via intravenous injection, we also find that active molecular targeting of the tumor microenvironments (e.g., fibroblasts, macrophages, and vasculatures) does not significantly influence the tumor nanoparticle uptake. These results suggest that for photothermal cancer therapy, the preferred route of gold nanorod administration is intra-tumoral injection instead of intravenous injection. PMID:20863096

Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity.

PubMed

Tasev, Dimitar; van Wijhe, Michiel H; Weijers, Ester M; van Hinsbergh, Victor W M; Koolwijk, Pieter

2015-01-01

Efficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs) as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs. The findings presented in this study indicate that human platelet lysate (PL) as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS). Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL) retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator) or uPAR (uPA receptor) by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential. The presented isolation method and subsequent cell expansion in platelet lysate supplemented culture medium permits suitable large-scale propagation of PB-ECFC. For optimal use of PB-ECFCs in clinical settings, our data suggest that 15-20 CPDL is the most adequate maturation stage.

Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity

PubMed Central

Tasev, Dimitar; van Wijhe, Michiel H.; Weijers, Ester M.; van Hinsbergh, Victor W. M.; Koolwijk, Pieter

2015-01-01

Introduction Efficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs) as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs. Results The findings presented in this study indicate that human platelet lysate (PL) as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS). Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL) retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator) or uPAR (uPA receptor) by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential. Conclusion The presented isolation method and subsequent cell expansion in platelet lysate supplemented culture medium permits suitable large-scale propagation of PB-ECFC. For optimal use of PB-ECFCs in clinical settings, our data suggest that 15–20 CPDL is the most adequate maturation stage. PMID:26076450

Anti-Metastatic and Anti-Tumor Growth Effects of Origanum majorana on Highly Metastatic Human Breast Cancer Cells: Inhibition of NFκB Signaling and Reduction of Nitric Oxide Production

PubMed Central

Al Dhaheri, Yusra; Attoub, Samir; Arafat, Kholoud; AbuQamar, Synan; Viallet, Jean; Saleh, Alaaeldin; Al Agha, Hala; Eid, Ali; Iratni, Rabah

2013-01-01

Background We have recently reported that Origanum majorana exhibits anticancer activity by promoting cell cycle arrest and apoptosis of the metastatic MDA-MB-231 breast cancer cell line. Here, we extended our study by investigating the effect of O . majorana on the migration, invasion and tumor growth of these cells. Results We demonstrate that non-cytotoxic concentrations of O . majorana significantly inhibited the migration and invasion of the MDA-MB-231 cells as shown by wound-healing and matrigel invasion assays. We also show that O . majorana induce homotypic aggregation of MDA-MB-231 associated with an upregulation of E-cadherin protein and promoter activity. Furthermore, we show that O . majorana decrease the adhesion of MDA-MB-231 to HUVECs and inhibits transendothelial migration of MDA-MB-231 through TNF-α-activated HUVECs. Gelatin zymography assay shows that O . majorana suppresses the activities of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9). ELISA, RT-PCR and Western blot results revealed that O . majorana decreases the expression of MMP-2, MMP-9, urokinase plasminogen activator receptor (uPAR), ICAM-1 and VEGF. Further investigation revealed that O . majorana suppresses the phosphorylation of IκB, downregulates the nuclear level of NFκB and reduces Nitric Oxide (NO) production in MDA-MB-231 cells. Most importantly, by using chick embryo tumor growth assay, we also show that O . majorana promotes inhibition of tumor growth and metastasis in vivo. Conclusion Our findings identify Origanum majorana as a promising chemopreventive and therapeutic candidate that modulate breast cancer growth and metastasis. PMID:23874773

Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator

PubMed Central

SAZONOVA, I. Y.; MCNAMEE, R. A.; HOUNG, A. K.; KING, S. M.; HEDSTROM, L.; REED, G. L.

2013-01-01

Summary Background: Given the worldwide epidemic of cardiovascular diseases, a more effective means of dissolving thrombi that cause heart attacks, could markedly reduce death, disability and healthcare costs. Plasminogen activators (PAs) such as streptokinase (SK) and tissue plasminogen activator (TPA) are currently used to dissolve fibrin thrombi. SK is cheaper and more widely available, but it appears less effective because it lacks TPA’s fibrin-targeted properties that focus plasminogen activation on the fibrin surface. Objective: We examined whether re-programming SK’s mechanism of action would create PAs with greater fibrin-targeting and potency than TPA. Methods and Results: When fibrinogen consumption was measured in human plasma, reprogrammed molecules SKΔ1 and SKΔ59 were 5-fold and > 119-fold more fibrin-dependent than SK (P < 0.0001), and 2-fold and > 50-fold more fibrin-dependent than TPA (P < 0.001). The marked fibrin-targeting of SKΔ59 was due to the fact that: (i) it did not generate plasmin in plasma, (ii) it was rapidly inhibited by α2-antiplasmin, and (iii) it only processed fibrin-bound plasminogen. To assess the fibrin-targeting and therapeutic potential of these PAs in vivo, a novel ‘humanized’ fibrinolysis model was created by reconstituting plasminogen-deficient mice with human plasminogen. When compared with TPA, SKΔ1 and SKΔ59 were 4-fold (P < 0.0001) and 2-fold (P < 0.003) more potent at dissolving blood clots in vivo, respectively, on a mass-dose basis and 2–3 logs more potent than TPA (P < 0.0001) when doses were calibrated by standard activity assays. Conclusion: These experiments suggest that reprogramming SK’s mechanism of action markedly enhances fibrin-targeting and creates, in comparison with TPA, activators with greater fibrinolytic potency. PMID:19566545

Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator.

PubMed

Sazonova, I Y; McNamee, R A; Houng, A K; King, S M; Hedstrom, L; Reed, G L

2009-08-01

Given the worldwide epidemic of cardiovascular diseases, a more effective means of dissolving thrombi that cause heart attacks, could markedly reduce death, disability and healthcare costs. Plasminogen activators (PAs) such as streptokinase (SK) and tissue plasminogen activator (TPA) are currently used to dissolve fibrin thrombi. SK is cheaper and more widely available, but it appears less effective because it lacks TPA's fibrin-targeted properties that focus plasminogen activation on the fibrin surface. We examined whether re-programming SK's mechanism of action would create PAs with greater fibrin-targeting and potency than TPA. When fibrinogen consumption was measured in human plasma, reprogrammed molecules SKDelta1 and SKDelta59 were 5-fold and > 119-fold more fibrin-dependent than SK (P < 0.0001), and 2-fold and > 50-fold more fibrin-dependent than TPA (P < 0.001). The marked fibrin-targeting of SKDelta59 was due to the fact that: (i) it did not generate plasmin in plasma, (ii) it was rapidly inhibited by alpha2-antiplasmin, and (iii) it only processed fibrin-bound plasminogen. To assess the fibrin-targeting and therapeutic potential of these PAs in vivo, a novel 'humanized' fibrinolysis model was created by reconstituting plasminogen-deficient mice with human plasminogen. When compared with TPA, SKDelta1 and SKDelta59 were 4-fold (P < 0.0001) and 2-fold (P < 0.003) more potent at dissolving blood clots in vivo, respectively, on a mass-dose basis and 2-3 logs more potent than TPA (P < 0.0001) when doses were calibrated by standard activity assays. These experiments suggest that reprogramming SK's mechanism of action markedly enhances fibrin-targeting and creates, in comparison with TPA, activators with greater fibrinolytic potency.

Antibodies Against Three Forms of Urokinase

NASA Technical Reports Server (NTRS)

Morrison, Dennis R.; Atassi, M. Zouhair

2007-01-01

Antibodies that bind to preselected regions of the urokinase molecule have been developed. These antibodies can be used to measure small quantities of each of three molecular forms of urokinase that could be contained in microsamples or conditioned media harvested from cultures of mammalian cells. Previously available antibodies and assay techniques do not yield both clear distinctions among, and measurements of, all three forms. Urokinase is a zymogen that is synthesized in a single-chain form, called ScuPA, which is composed of 411 amino acid residues (see figure). ScuPA has very little enzyme activity, but it can be activated in two ways: (1) by cleavage of the peptide bond lysine 158/isoleucine 159 and the loss of lysine 158 to obtain the high molecular-weight (HMW) form of the enzyme or (2) by cleavage of the bond lysine 135/lysine 136 to obtain the low-molecular-weight (LMW) form of the enzyme. The antibodies in question were produced in mice and rabbits by use of peptides as immunogens. The peptides were selected to obtain antibodies that bind to regions of ScuPA that include the lysine 158/isoleucine 159 and the lysine 135/lysine 136 bonds. The antibodies include monoclonal and polyclonal ones that yield indications as to whether either of these bonds is intact. The polyclonal antibodies include ones that preferentially bind to the HMW or LMW forms of the urokinase molecule. The monoclonal antibodies include ones that discriminate between the ScuPA and the HMW form. A combination of these molecular-specific antibodies will enable simultaneous assays of the ScuPA, HMW, and LMW forms in the same specimen of culture medium.

Polyphenol fatty acid esters as serine protease inhibitors: a quantum-chemical QSAR analysis.

PubMed

Viskupicova, Jana; Danihelova, Martina; Majekova, Magdalena; Liptaj, Tibor; Sturdik, Ernest

2012-12-01

We investigated the ability of polyphenol fatty acid esters to inhibit the activity of serine proteases trypsin, thrombin, elastase and urokinase. Potent protease inhibition in micromolar range was displayed by rutin and rutin derivatives esterified with medium and long chain, mono- and polyunsaturated fatty acids (1e-m), followed by phloridzin and esculin esters with medium and long fatty acid chain length (2a-d, 3a-d), while unmodified compounds showed only little or no effect. QSAR study of the compounds tested provided the most significant parameters for individual inhibition activities, i.e. number of hydrogen bond donors for urokinase, molecular volume for thrombin, and solvation energy for elastase. According to the statistical analysis, the action of elastase inhibitors is opposed to those of urokinase and thrombin. Cluster analysis showed two groups of compounds: original polyphenols together with rutin esters with short fatty acid chain length and rutin esters with long fatty acid chain length.

Efficient copackaging and cotransport yields postsynaptic colocalization of neuromodulators associated with synaptic plasticity.

PubMed

Lochner, J E; Spangler, E; Chavarha, M; Jacobs, C; McAllister, K; Schuttner, L C; Scalettar, B A

2008-09-01

Recent data suggest that tissue plasminogen activator (tPA) influences long-term plasticity at hippocampal synapses by converting plasminogen into plasmin, which then generates mature brain-derived neurotrophic factor (mBDNF) from its precursor, proBDNF. Motivated by this hypothesis, we used fluorescent chimeras, expressed in hippocampal neurons, to elucidate (1) mechanisms underlying plasminogen secretion from hippocampal neurons, (2) if tPA, plasminogen, and proBDNF are copackaged and cotransported in hippocampal neurons, especially within dendritic spines, and (3) mechanisms mediating the transport of these neuromodulators to sites of release. We find that plasminogen chimeras traffic through the regulated secretory pathway of hippocampal neurons in dense-core granules (DCGs) and that tPA, plasminogen, and proBDNF chimeras are extensively copackaged in DCGs throughout hippocampal neurons. We also find that 80% of spines that contain DCGs contain chimeras of these neuromodulators in the same DCG. Finally, we demonstrate, for the first time, that neuromodulators undergo cotransport along dendrites in rapidly mobile DCGs, indicating that neuromodulators can be efficiently recruited into active spines. These results support the hypothesis that tPA mediates synaptic activation of BDNF by demonstrating that tPA, plasminogen, and proBDNF colocalize in DCGs in spines, where these neuromodulators can undergo activity-dependent release and then interact and/or mediate changes that influence synaptic efficacy. The results also raise the possibility that frequency-dependent changes in extents of neuromodulator release from DCGs influence the direction of plasticity at hippocampal synapses by altering the relative proportions of two proteins, mBDNF and proBDNF, that exert opposing effects on synaptic efficacy.

Expression of PRSS, the plasminogen activator system and its activity in the ovine placentome during Stage 2 of parturition

USDA-ARS?s Scientific Manuscript database

The molecular mechanisms responsible for placenta separation are not completely understood. We know placentomes from cases of retained placenta possess limited matrix-metalloprotease (MMP) activity and retained placenta occurs at a greater incidence during induced parturition. The plasminogen activ...

Overexpression of regulator of G protein signaling 11 promotes cell migration and associates with advanced stages and aggressiveness of lung adenocarcinoma.

PubMed

Yang, Sheng-Huei; Li, Chien-Feng; Chu, Pei-Yi; Ko, Hsiu-Hsing; Chen, Li-Tzong; Chen, Wan-Wen; Han, Chia-Hung; Lung, Jr-Hau; Shih, Neng-Yao

2016-05-24

Regulator of G protein signaling 11 (RGS11), a member of the R7 subfamily of RGS proteins, is a well-characterized GTPase-accelerating protein that is involved in the heterotrimeric G protein regulation of the amplitude and kinetics of receptor-promoted signaling in retinal bipolar and nerve cells. However, the role of RGS11 in cancer is completely unclear. Using subtractive hybridization analysis, we found that RGS11 was highly expressed in the lymph-node metastatic tissues and bone-metastatic tumors obtained from patients with lung adenocarcinoma. Characterization of the clinicopathological features of 91 patients showed that around 57.1% of the tumor samples displayed RGS11 overexpression that was associated with primary tumor status, nodal metastasis and increased disease stages. Its high expression was an independent predictive factor for poor prognosis of these patients. Cotransfection of guanine nucleotide-binding protein beta-5 (GNB5) markedly increased RGS11 expression. Enhancement or attenuation of RGS11 expression pinpointed its specific role in cell migration, but not in cell invasion and proliferation. Signaling events initiated by the RGS11-GNB5 coexpression activated the c-Raf/ERK/FAK-mediated pathway through upregulation of the Rac1 activity. Consistently, increasing the cell invasiveness of the transfectants by additional cotransfection of the exogenous urokinase-plasminogen activator gene caused a significant promotion in cell invasion in vitro and in vivo, confirming that RGS11 functions in cell migration, but requires additional proteolytic activity for cell and tissue invasion. Collectively, overexpression of RGS11 promotes cell migration, participates in tumor metastasis, and correlates the clinicopathological conditions of patients with lung adenocarcinoma.

Influence of natural humic acids and synthetic phenolic polymers on fibrinolysis

NASA Astrophysics Data System (ADS)

Klöcking, Hans-Peter

The influence of synthetic and natural phenolic polymers on the release of plasminogen activator was studied in an isolated, perfused, vascular preparation (pig ear). Of the tested synthetic phenolic polymers, the oxidation products of caffeic acid (KOP) and 3,4-dihydroxyphenylacetic acid (3,4-DHPOP), at a concentration of 50 µg/ml perfusate, were able to increase the plasminogen activator activity by 70%. The oxidation products of chlorogenic acid (CHOP), hydrocaffeic acid (HYKOP), pyrogallol (PYROP) and gallic acid (GALOP), at the same concentration, exerted no influence on the release of plasminogen activator. Of the naturally occurring humic acids, the influence of sodium humate was within the same order of magnitude as KOP and 3,4-DHPOP. Ammonium humate was able to increase the plasminogen activator release only at a concentration of 100 µg/ml perfusate. In rats, the t-PA activity increased after i.v. application of 10 mg/kg of KOP, Na-HS or NH4-HS.

A study of the possible association of plasminogen activator inhibitor type 1 4G/5G insertion/deletion polymorphism with susceptibility to schizophrenia and in its subtypes.

PubMed

Yenilmez, C; Ozdemir Koroglu, Z; Kurt, H; Yanas, M; Colak, E; Degirmenci, I; Gunes, H V

2017-02-01

Inhibition of the fibrinolytic system may occur at the level of plasminogen activation, mainly by PAI-1. Mental and physical stress caused to alterations of platelet function, and also decreased to fibrinolytic activity. Furthermore, stress-induced thrombosis regulation was proposed to be by PAI-1 in schizophrenia patients. In this study, the distribution of genotypes and frequency of alleles of the plasminogen activator inhibitor type 1 (PAI-1) gene 4G/5G polymorphism in different Turkish clinical schizophrenia subtypes was investigated for its role in schizophrenia development. The clinical schizophrenia subtypes include paranoid, catatonic, disorganized, undifferentiated and residual, as diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition IV (DSM-IV). Samples of genomic DNA (250 total, including 150 schizophrenia patients and 100 healthy subjects) were analysed. PAI-1 4G/5G genotyping was performed by polymerase chain reaction-allele-specific amplification. PCR products were separated by 2% agarose gel electrophoresis and then visualized. The genotype distributions (P = 0·136) and allele frequencies (P = 0·721 for 4G, P = 0. 097 for 5G) were not significantly different between patients with schizophrenia and control subjects for the 4G/5G polymorphism. Similar results were also found for the genotype distributions (P = 0·640) and allele frequencies (P = 0·763 for 4G, P = 0·448 for 5G) in the clinical schizophrenia subtypes compared to the each other. We conclude that PAI-1 4G/5G polymorphism was not significantly associated with schizophrenia or its subtypes in the Turkish population. However, we recognize that with our sample sizes, we cannot exclude weak associations. © 2016 John Wiley & Sons Ltd.

Adsorption properties for urokinase on local diatomite surface

NASA Astrophysics Data System (ADS)

Yang, Yuxiang; Zhang, Jianbo; Yang, Weimin; Wu, Jieda; Chen, Rongsan

2003-02-01

In this paper, adsorption isotherm of urokinase on two typical local diatomites were determined at 25 °C and their surface electrical potentials (ζ), isoelectrical point values (IEP) were determined. The properties of diatomites, the relationship among diatomite structure, pore-size distribution, surface ζ and adsorption isotherm were discussed. The adsorption equation of urokinase was calculated from the adsorption isotherm. The adsorption mode of urokinase on diatomite surface was judged by the configuration function α. The relationship between the amount of adsorbed urokinase and IEP value was also discussed.

The Serine Protease Inhibitor Neuroserpin Is Required for Normal Synaptic Plasticity and Regulates Learning and Social Behavior

ERIC Educational Resources Information Center

Reumann, Rebecca; Vierk, Ricardo; Zhou, Lepu; Gries, Frederice; Kraus, Vanessa; Mienert, Julia; Romswinkel, Eva; Morellini, Fabio; Ferrer, Isidre; Nicolini, Chiara; Fahnestock, Margaret; Rune, Gabriele; Glatzel, Markus; Galliciotti, Giovanna

2017-01-01

The serine protease inhibitor neuroserpin regulates the activity of tissue-type plasminogen activator (tPA) in the nervous system. Neuroserpin expression is particularly prominent at late stages of neuronal development in most regions of the central nervous system (CNS), whereas it is restricted to regions related to learning and memory in the…

Peroxisome Proliferator-Activated Receptor-γ Ligands Alter Breast Cancer Cell Motility through Modulation of the Plasminogen Activator System

PubMed Central

Carter, Jennifer C.; Church, Frank C.

2011-01-01

We investigated peroxisome proliferator-activated receptor-γ (PPAR-γ) ligands effect on cell motility and the plasminogen activator system using normal MCF-10A and malignant MCF-10CA1 cell lines. Ciglitazone reduced both wound-induced migration and chemotaxis. However, the effect was not reversed with pretreatment of cells with the PPAR-γ-specific antagonist GW9662. Immunoblot analysis of conditioned media showed ciglitazone decreased plasminogen activator inhibitor-1 (PAI-1) in both cell lines; this effect was also unaltered by PPAR-γ antagonism. Alternatively, treatment with the ω-6 fatty acid arachidonic acid (ArA), but not the ω-3 fatty acid docosahexanoic acid, increased both MCF-10A cell migration and cell surface uPA activity. Pretreatment with a PPAR-γ antagonist reversed these effects, suggesting that ArA mediates its effect on cell motility and uPA activity through PPAR-γ activation. Collectively, the data suggest PPAR-γ ligands have a differential effect on normal and malignant cell migration and the plasminogen activation system, resulting from PPAR-γ-dependent and PPAR-γ-independent effects. PMID:22131991

[In vitro function of outer membrane protease T of Escherichia coli K1 pathogenic strain].

PubMed

Hui, Changye; Guo, Yan; Wu, Shuchi; Peng, Liang; Cao, Hong; Huang, Shenghe

2010-01-01

Plasminogen activation and antimicrobial peptide hydrolysis contribute to pathogens invasion and survival in vivo. To demonstrate the expression of outer membrane protease T in E. coli K1 pathogenic strain E44, its activity of plasminogen activator and protamine hydrolysis. After Benzamidine Sepharose Fast Flow and SOURCE 30Q chromatography, we got E44 outer membrane mixed fraction, and examined its activity of plasminogen activation with chromogenic substrate S-2251 method. An ompT deletion mutant of E44 was constructed by using the suicide vector pCVD442, termed as E44ompT. We examined 0.1 mg/mL cationic antimicrobial peptide protamine susceptibility of E44, ompT mutant strain E44ompT and E44ompT harboring pUCT, which was constructed by inserting complete ompT open reading frame into pUC13. We got about 37 kDa E44 membrane extract, which could activate plasminogen, and activation was membrane extract dose dependent. This confirmed the expression of outer membrane protease T in the outer membrane of E44. E44ompT was, more susceptible to 0.1 mg/mL protamine than E44, and E440mpT was partially complemented by pUCT. Outer membrane protease T is expressed in E. coli K1 pathogenic strain E44, and can activate plasminogen and hydrolyze protamine.

Plasmin-dependent modulation of the blood–brain barrier: a major consideration during tPA-induced thrombolysis?

PubMed Central

Niego, Be'eri; Medcalf, Robert L

2014-01-01

Plasmin, the principal downstream product of tissue-type plasminogen activator (tPA), is known for its potent fibrin-degrading capacity but is also recognized for many non-fibrinolytic activities. Curiously, plasmin has not been conclusively linked to blood–brain barrier (BBB) disruption during recombinant tPA (rtPA)-induced thrombolysis in ischemic stroke. This is surprising given the substantial involvement of tPA in the modulation of BBB permeability and the co-existence of tPA and plasminogen in both blood and brain throughout the ischemic event. Here, we review the work that argues a role for plasmin together with endogenous tPA or rtPA in BBB alteration, presenting the overall controversy around the topic yet creating a rational case for an involvement of plasmin in this process. PMID:24896566

Analysis of Paracoccidioides secreted proteins reveals fructose 1,6-bisphosphate aldolase as a plasminogen-binding protein.

PubMed

Chaves, Edilânia Gomes Araújo; Weber, Simone Schneider; Báo, Sonia Nair; Pereira, Luiz Augusto; Bailão, Alexandre Melo; Borges, Clayton Luiz; Soares, Célia Maria de Almeida

2015-02-27

Despite being important thermal dimorphic fungi causing Paracoccidioidomycosis, the pathogenic mechanisms that underlie the genus Paracoccidioides remain largely unknown. Microbial pathogens express molecules that can interact with human plasminogen, a protein from blood plasma, which presents fibrinolytic activity when activated into plasmin. Additionally, plasmin exhibits the ability of degrading extracellular matrix components, favoring the pathogen spread to deeper tissues. Previous work from our group demonstrated that Paracoccidioides presents enolase, as a protein able to bind and activate plasminogen, increasing the fibrinolytic activity of the pathogen, and the potential for adhesion and invasion of the fungus to host cells. By using proteomic analysis, we aimed to identify other proteins of Paracoccidioides with the ability of binding to plasminogen. In the present study, we employed proteomic analysis of the secretome, in order to identify plasminogen-binding proteins of Paracoccidioides, Pb01. Fifteen proteins were present in the fungal secretome, presenting the ability to bind to plasminogen. Those proteins are probable targets of the fungus interaction with the host; thus, they could contribute to the invasiveness of the fungus. For validation tests, we selected the protein fructose 1,6-bisphosphate aldolase (FBA), described in other pathogens as a plasminogen-binding protein. The protein FBA at the fungus surface and the recombinant FBA (rFBA) bound human plasminogen and promoted its conversion to plasmin, potentially increasing the fibrinolytic capacity of the fungus, as demonstrated in fibrin degradation assays. The addition of rFBA or anti-rFBA antibodies was capable of reducing the interaction between macrophages and Paracoccidioides, possibly by blocking the binding sites for FBA. These data reveal the possible participation of the FBA in the processes of cell adhesion and tissue invasion/dissemination of Paracoccidioides. These data indicate that Paracoccidioides is a pathogen that has several plasminogen-binding proteins that likely play important roles in pathogen-host interaction. In this context, FBA is a protein that might be involved somehow in the processes of invasion and spread of the fungus during infection.

Examination of the signal transduction pathways leading to upregulation of tissue type plasminogen activator by Porphyromonas endodontalis in human pulp cells.

PubMed

Huang, F-M; Chen, Y-J; Chou, M-Y; Chang, Y-C

2005-12-01

To investigate the tissue type plasminogen activator (t-PA) activity in human pulp cells stimulated with Porphyromonas endodontalis (P. endodontalis) in the absence or presence of p38 inhibitor SB203580, mitogen-activated protein kinase kinase (MEK) inhibitor U0126 and phosphatidylinositaol 3-kinase (PI3K) inhibitor LY294002. The supernatants of P. endodontalis were used to evaluate t-PA activity in human pulp cells using casein zymography and enzyme-linked immunosorbent assay (ELISA). Furthermore, to search for possible signal transduction pathways, SB203580, U0126 and LY294002 were added to test how they modulated the t-PA activity. The main casein secreted by human pulp cells migrated at 70 kDa and represented t-PA. Secretion of t-PA was found to be stimulated with P. endodontalis during 2-day cultured period (P < 0.05). From the results of casein zymography and ELISA, SB203580 and U0126 significantly reduced the P. endodontalis stimulated t-PA production respectively (P < 0.05). However, LY294002 lacked the ability to change the P. endodontalis stimulated t-PA production (P > 0.05). Porphyromonas endodontalis enhances t-PA production in human pulp cells, and the signal transduction pathways p38 and MEK are involved in the inhibition of t-PA.

Efficient co-packaging and co-transport yields post-synaptic co-localization of neuromodulators associated with synaptic plasticity

PubMed Central

Lochner, J. E.; Spangler, E.; Chavarha, M.; Jacobs, C.; McAllister, K.; Schuttner, L. C.; Scalettar, B. A.

2009-01-01

Recent data suggest that tissue plasminogen activator (tPA) influences long-term plasticity at hippocampal synapses by converting plasminogen into plasmin, which then generates mature brain-derived neurotrophic factor (mBDNF) from its precursor, proBDNF. Motivated by this hypothesis, we used fluorescent chimeras, expressed in hippocampal neurons, to elucidate (1) mechanisms underlying plasminogen secretion from hippocampal neurons, (2) if tPA, plasminogen, and proBDNF are co-packaged and co-transported in hippocampal neurons, especially within dendritic spines, and (3) mechanisms mediating the transport of these neuromodulators to sites of release. We find that plasminogen chimeras traffic through the regulated secretory pathway of hippocampal neurons in dense-core granules (DCGs) and that tPA, plasminogen, and proBDNF chimeras are extensively co-packaged in DCGs throughout hippocampal neurons. We also find that 80% of spines that contain DCGs contain chimeras of these neuromodulators in the same DCG. Finally, we demonstrate, for the first time, that neuromodulators undergo co-transport along dendrites in rapidly mobile DCGs, indicating that neuromodulators can be efficiently recruited into active spines. These results support the hypothesis that tPA mediates synaptic activation of BDNF by demonstrating that tPA, plasminogen, and proBDNF co-localize in DCGs in spines, where these neuromodulators can undergo activity-dependent release and then interact and/or mediate changes that influence synaptic efficacy. The results also raise the possibility that frequency-dependent changes in extents of neuromodulator release from DCGs influence the direction of plasticity at hippocampal synapses by altering the relative proportions of two proteins, mBDNF and proBDNF, that exert opposing effects on synaptic efficacy. PMID:18563704

Stimulation of plasmin activity in cultured human fibroblast cells by Porphyromonas endodontalis.

PubMed

Oikawa, T; Ogura, N; Akiba, M; Abiko, Y; Takiguchi, H; Izumi, H

1993-09-01

1. Plasmin activity in the conditioned medium of Gin-1 cells, a human gingival fibroblast cell line, was stimulated by Porphyromonas endodontalis, a putative pathogen of oral submucous abscesses, in a time- and dose-dependent manner. 2. P. endodontalis stimulated the activity of plasminogen activator in both the conditioned medium and the cell lysate. The plasminogen activator in Gin-1 cells was approx. 50 kDa by zymography. 3. The conditioned medium of Gin-1 cells exposed to P. endodontalis stimulated the conversion of human serum prekallikrein to kallikrein. 4. These results suggested that P. endodontalis stimulates the plasminogen activator-plasmin system in Gin-1 cells, and that activated plasmin plays a role in the progress of periodontal tissue inflammation.

Thrombin-activable fibrinolysis inhibitor attenuates (DD)E-mediated stimulation of plasminogen activation by reducing the affinity of (DD)E for tissue plasminogen activator. A potential mechanism for enhancing the fibrin specificity of tissue plasminogen activator.

PubMed

Stewart, R J; Fredenburgh, J C; Rischke, J A; Bajzar, L; Weitz, J I

2000-11-24

A complex of d-dimer noncovalently associated with fragment E ((DD)E), a degradation product of cross-linked fibrin that binds tissue plasminogen activator (t-PA) and plasminogen (Pg) with affinities similar to those of fibrin, compromises the fibrin specificity of t-PA by stimulating systemic Pg activation. In this study, we examined the effect of thrombin-activable fibrinolysis inhibitor (TAFI), a latent carboxypeptidase B (CPB)-like enzyme, on the stimulatory activity of (DD)E. Incubation of (DD)E with activated TAFI (TAFIa) or CPB (a) produces a 96% reduction in the capacity of (DD)E to stimulate t-PA-mediated activation of Glu- or Lys-Pg by reducing k(cat) and increasing K(m) for the reaction; (b) induces the release of 8 mol of lysine/mol of (DD)E, although most of the stimulatory activity is lost after release of only 4 mol of lysine/mol (DD)E; and (c) reduces the affinity of (DD)E for Glu-Pg, Lys-Pg, and t-PA by 2-, 4-, and 160-fold, respectively. Because TAFIa- or CPB-exposed (DD)E produces little stimulation of Glu-Pg activation by t-PA, (DD)E is not degraded into fragment E and d-dimer, the latter of which has been reported to impair fibrin polymerization. These data suggest a novel role for TAFIa. By attenuating systemic Pg activation by (DD)E, TAFIa renders t-PA more fibrin-specific.

Randomized comparison of intracoronary tirofiban versus urokinase as an adjunct to primary percutaneous coronary intervention in patients with acute ST-elevation myocardial infarction: results of the ICTUS-AMI trial.

PubMed

Zhu, Tian-qi; Zhang, Qi; Ding, Feng-hua; Qiu, Jian-ping; Jin, Hui-geng; Jiang, Li; Lu, Lin; Zhang, Rui-yan; Hu, Jian; Yang, Zhen-kun; Shen, Ying; Shen, Wei-feng

2013-08-01

No randomized trial has been performed to compare the efficacy of an intracoronary bolus of tirofiban versus urokinase during primary percutaneous coronary intervention (PCI). We investigated whether the effects of adjunctive therapy with an intracoronary bolus of urokinase was noninferior to the effects of an intracoronary bolus of tirofiban in patients with ST-elevation myocardial infarction (STEMI) undergoing PCI. A total of 490 patients with acute STEMI undergoing primary PCI were randomized to an intracoronary bolus of tirofiban (10 µg/kg; n = 247) or urokinase (250 kU/20 ml; n = 243). Serum levels of P-selectin, von Willebrand factor (vWF), CD40 ligand (CD40L), and serum amyloid A (SAA) in the coronary sinus were measured before and after intracoronary drug administration. The primary endpoint was the rate of complete ( ≥ 70%) ST-segment resolution (STR) at 90 minutes after intervention, and the noninferiority margin was set to 15%. In the intention-to-treat analysis, complete STR was achieved in 54.4% of patients treated with an intracoronary bolus of urokinase and in 60.6% of those treated with an intracoronary bolus of tirofiban (adjusted difference: -7.0%; 95% confidence interval: -15.7% to 1.8%). The corrected TIMI frame count of the infarct-related artery was lower, left ventricular ejection fraction was higher, and the 6-month major adverse cardiac event-free survival tended to be better in the intracoronary tirofiban group. An intracoronary bolus of tirofiban resulted in lower levels of P-selectin, vWF, CD40L, and SAA in the coronary sinus compared with an intracoronary bolus of urokinase after primary PCI (P < 0.05). An intracoronary bolus of urokinase as an adjunct to primary PCI for acute STEMI is not equally effective to an intracoronary bolus of tirofiban with respect to improvement in myocardial reperfusion assessed by STR. This may be caused by less reduction in coronary circulatory platelet activation and inflammation.

Evaluation of Prognostic Values of Tissue Plasminogen Activator and Plasminogen Activator Inhibitor-1 in Crimean-Congo Hemorrhagic Fever Patients

PubMed Central

Gurbuz, Yunus; Ozturk, Baris; Tutuncu, Emin Ediz; Sencan, Irfan; Cicek Senturk, Gonul; Altay, Fatma Aybala

2015-01-01

Background: Crimean-Congo hemorrhagic fever (CCHF) is a widespread disease in Turkey, and was responsible for many deaths in endemic regions during the last decade. The pathogenesis of the disease is not fully understood yet. Objectives: In this study we aimed to determine the levels of tissue plasminogen activator (tPA) and Plasminogen activator inhibitor-1 (PAI-1) as predictors of prognosis in CCHF. Patients and Methods: Patients who were diagnosed by the polymerase chain reaction (PCR) and IgM positivity in the reference laboratory were included in this study. Tissue Plasminogen activator and PAI-1 levels were measured by the enzyme linked immunosorbent assay (ELISA) using a commercial kit (human t-PA ELISA and human PAL-1 ELISA; BioVendor research and diagnostic products, BioVendor-Laboratorni medicina a.s., Brno, Czech Republic). Results: A total of 46 patients participated in this study. The significant differences between recovering patients and the patients who died, regarding Aspartate aminotransferase (AST), Creatine Phosphokinase (CPK), Lactate Dehydrogenase (LDH), Prothrombin Time (PT), activated Partial Thromboplastin time (aPTT), and thrombocyte and fibrinogen levels, were consistent with many clinical studies in the literature. The fatal cases were found to have higher tPA and PAI-1 levels in contrast to the patients who completely recovered. Conclusions: We think that these findings may help the progress of understanding of CCHF pathogenesis. PMID:26587219

Comparative toxicity and efficacy of engineered anthrax lethal toxin variants with broad anti-tumor activities.

PubMed

Peters, Diane E; Hoover, Benjamin; Cloud, Loretta Grey; Liu, Shihui; Molinolo, Alfredo A; Leppla, Stephen H; Bugge, Thomas H

2014-09-01

We have previously designed and characterized versions of anthrax lethal toxin that are selectively cytotoxic in the tumor microenvironment and which display broad and potent anti-tumor activities in vivo. Here, we have performed the first direct comparison of the safety and efficacy of three engineered anthrax lethal toxin variants requiring activation by either matrix-metalloproteinases (MMPs), urokinase plasminogen activator (uPA) or co-localized MMP/uPA activities. C57BL/6J mice were challenged with six doses of engineered toxins via intraperitoneal (I.P.) or intravenous (I.V.) dose routes to determine the maximum tolerated dose for six administrations (MTD6) and dose-limiting toxicities. Efficacy was evaluated using the B16-BL6 syngraft model of melanoma; mice bearing established tumors were treated with six I.P. doses of toxin and tumor measurements and immunohistochemistry, paired with terminal blood work, were used to elaborate upon the anti-tumor mechanism and relative efficacy of each variant. We found that MMP-, uPA- and dual MMP/uPA-activated anthrax lethal toxins exhibited the same dose-limiting toxicity; dose-dependent GI toxicity. In terms of efficacy, all three toxins significantly reduced primary B16-BL6 tumor burden, ranging from 32% to 87% reduction, and they also delayed disease progression as evidenced by dose-dependent normalization of blood work values. While target organ toxicity and effective doses were similar amongst the variants, the dual MMP/uPA-activated anthrax lethal toxin exhibited the highest I.P. MTD6 and was 1.5-3-fold better tolerated than the single MMP- and uPA-activated toxins. Overall, we demonstrate that this dual MMP/uPA-activated anthrax lethal toxin can be administered safely and is highly effective in a preclinical model of melanoma. This modified bacterial cytotoxin is thus a promising candidate for further clinical development and evaluation for use in treating human cancers. Published by Elsevier Inc.

Comparative toxicity and efficacy of engineered anthrax lethal toxin variants with broad anti-tumor activities

PubMed Central

Peters, Diane E.; Hoover, Benjamin; Cloud, Loretta Grey; Liu, Shihui; Molinolo, Alfredo A.; Leppla, Stephen H.; Bugge, Thomas H.

2014-01-01

We have previously designed and characterized versions of anthrax lethal toxin that are selectively cytotoxic in the tumor microenvironment and which display broad and potent anti-tumor activities in vivo. Here, we have performed the first direct comparison of the safety and efficacy of three engineered anthrax lethal toxin variants requiring activation by either matrix-metalloproteinases (MMPs), urokinase plasminogen activator (uPA) or co-localized MMP/uPA activities. C57BL/6J mice were challenged with six doses of engineered toxins via intraperitoneal (I.P.) or intravenous (I.V.) dose routes to determine the maximum tolerated dose for six administrations (MTD6) and dose-limiting toxicities. Efficacy was evaluated using the B16-BL6 syngraft model of melanoma; Mice bearing established tumors were treated with six I.P. doses of toxin and tumor measurements and immunohistochemistry, paired with terminal blood work, were used to elaborate upon the anti-tumor mechanism and relative efficacy of each variant. We found that MMP-, uPA- and dual MMP/uPA- activated anthrax lethal toxins exhibited the same dose-limiting toxicity; dose-dependent GI toxicity. In terms of efficacy, all three toxins significantly reduced primary B16-BL6 tumor burden, ranging from 32%–87% reduction, and they also delayed disease progression as evidenced by dose-dependent normalization of blood work values. While target organ toxicity and effective doses were similar amongst the variants, the dual MMP/uPA-activated anthrax lethal toxin exhibited the highest I.P. MTD6 and was 1.5–3-fold better tolerated than the single MMP- and uPA-activated toxins. Overall, we demonstrate that this dual MMP/uPA-activated anthrax lethal toxin can be administered safely and is highly effective in a preclinical model of melanoma. This modified bacterial cytotoxin is thus a promising candidate for further clinical development and evaluation for use in treating human cancers. PMID:24971906

Quercetin Has Antimetastatic Effects on Gastric Cancer Cells via the Interruption of uPA/uPAR Function by Modulating NF-κb, PKC-δ, ERK1/2, and AMPKα.

PubMed

Li, Hai; Chen, Chen

2018-06-01

Gastric cancer (GC) is a malignancy with few effective treatment options after metastasis occurs. Quercetin (Qu) intake has been associated with reduced incidence and slow development of GC, probably due to its anti-proliferative and apoptotic effects, but it is unclear whether Qu can inhibit the metastatic activity. The urokinase plasminogen activator (uPA)/uPA receptor (uPAR) system plays an important role in cancer metastasis. In this study, we measured both uPA activity and uPAR expression in GC and pericarcinous tissues, and we investigated the correlation between uPAR expression and the migratory and invasive activities of various GC cell lines. GC BGC823 and AGS cells were subjected to treatment with 10 μM Qu for 72 hours and uPAR knockdown, alone or in combination, before evaluating cell metastasis. The results showed that uPA activity and uPAR expression were higher in GC tissues than in pericarcinous tissues. Migratory and invasive activities of GC cell lines positively correlated with uPAR expression. Qu treatment decreased BGC823 and AGS cell migration and invasion, accompanied by reduced uPA and uPAR protein expression. Both Qu treatment and uPAR knockdown decreased matrix metalloproteinase-2 and -9 activity and blocked Pak1-Limk1-cofilin signaling. Qu treatment was associated with inhibition of NF-κb, PKC-δ, and ERK1/2, and with AMPKα activation. Specific inhibitors of NF-κb, PKC, and ERK1/2, and an AMPKα activator suppressed uPA and uPAR expression in GC cells. Collectively, Qu showed an antimetastatic effect on GC cells via the interruption of uPA/uPAR function and modulation of NF-κb, PKC-δ, ERK1/2, and AMPKα. This suggests that Qu is a promising agent against GC metastasis.

Biochemical actions of glucocorticoids on macrophages in culture. Specific inhibition of elastase, collagenase, and plasminogen activator secretion and effects on other metabolic functions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Werb, Z.

1978-01-01

The effects of glucocorticoids on biochemical functions of macrophages from man, mouse, rabbit, and guinea pig were examined. Secretion of plasminogen activator by human peripheral blood monocytes was decreased 50% with 1 nM dexamethasone. Differentiation of murine monocytic and granulocytic colonies in agar from bone marrow precursors was decreased 50% at 7 days with 20 nM dexamethasone. Secretion of elastase, collagenase, and plasminogen activator by resident and thioglycollate-elicited mouse peritoneal macrophages was decreased by dexamethasone, cortisol, and triamcinolone acetonide (1 to 1,000 nM), but not by progesterone, estradiol, and dihydrotestosterone (1,000 nM); in contast, secretion of lysozyme was not affectedmore » by glucocorticoids. The inhibition of macrophage secretion by dexamethasone was both time and dose dependent. Inhibition of macrophage secretion increased with increasing glucocorticoid concentration. Half-maximum inhibition of secretion of elastase, collagenase, and plasminogen activator was seen at dexamethasone concentrations (1 to 10 nM) similar to those that half-saturated the specific glucocorticoid receptors. At high concentrations of dexamethasone (100 to 1,000 nM) the secretion of plasminogen activator was inhibited to a greater extent (>95%) than the secretion of elastase (60 to 80%).Progesterone alone had no effect on secretion, but blocked the inhibitory effects of dexamethasone and cortisol. Secretion of collagenase, neutral proteinases, and plasminogen activator by elicited rabbit alveolar macrophages was inhibited with glucocorticoids (0.1 to 100 nM) but not with progesterone or sex steroids. Secretion of a neutral elastinolytic proteinase by guinea pig alveolar macrophages was also inhibited by dexamethasone.« less

A collaborative sequential meta-analysis of individual patient data from randomized trials of endovascular therapy and tPA vs. tPA alone for acute ischemic stroke: ThRombEctomy And tPA (TREAT) analysis: statistical analysis plan for a sequential meta-analysis performed within the VISTA-Endovascular collaboration.

PubMed

MacIsaac, Rachael L; Khatri, Pooja; Bendszus, Martin; Bracard, Serge; Broderick, Joseph; Campbell, Bruce; Ciccone, Alfonso; Dávalos, Antoni; Davis, Stephen M; Demchuk, Andrew; Diener, Hans-Christoph; Dippel, Diederik; Donnan, Geoffrey A; Fiehler, Jens; Fiorella, David; Goyal, Mayank; Hacke, Werner; Hill, Michael D; Jahan, Reza; Jauch, Edward; Jovin, Tudor; Kidwell, Chelsea S; Liebeskind, David; Majoie, Charles B; Martins, Sheila Cristina Ouriques; Mitchell, Peter; Mocco, J; Muir, Keith W; Nogueira, Raul; Saver, Jeffrey L; Schonewille, Wouter J; Siddiqui, Adnan H; Thomalla, Götz; Tomsick, Thomas A; Turk, Aquilla S; White, Philip; Zaidat, Osama; Lees, Kennedy R

2015-10-01

Endovascular treatment has been shown to restore blood flow effectively. Second-generation medical devices such as stent retrievers are now showing overwhelming efficacy in clinical trials, particularly in conjunction with intravenous recombinant tissue plasminogen activator. This statistical analysis plan utilizing a novel, sequential approach describes a prospective, individual patient data analysis of endovascular therapy in conjunction with intravenous recombinant tissue plasminogen activator agreed upon by the Thrombectomy and Tissue Plasminogen Activator Collaborative Group. This protocol will specify the primary outcome for efficacy, as 'favorable' outcome defined by the ordinal distribution of the modified Rankin Scale measured at three-months poststroke, but with modified Rankin Scales 5 and 6 collapsed into a single category. The primary analysis will aim to answer the questions: 'what is the treatment effect of endovascular therapy with intravenous recombinant tissue plasminogen activator compared to intravenous tissue plasminogen activator alone on full scale modified Rankin Scale at 3 months?' and 'to what extent do key patient characteristics influence the treatment effect of endovascular therapy?'. Key secondary outcomes include effect of endovascular therapy on death within 90 days; analyses of modified Rankin Scale using dichotomized methods; and effects of endovascular therapy on symptomatic intracranial hemorrhage. Several secondary analyses will be considered as well as expanding patient cohorts to intravenous recombinant tissue plasminogen activator-ineligible patients, should data allow. This collaborative meta-analysis of individual participant data from randomized trials of endovascular therapy vs. control in conjunction with intravenous thrombolysis will demonstrate the efficacy and generalizability of endovascular therapy with intravenous thrombolysis as a concomitant medication. © 2015 World Stroke Organization.

Quantitative method of measuring cancer cell urokinase and metastatic potential

NASA Technical Reports Server (NTRS)

Morrison, Dennis R. (Inventor)

1993-01-01

The metastatic potential of tumors can be evaluated by the quantitative detection of urokinase and DNA. The cell sample selected for examination is analyzed for the presence of high levels of urokinase and abnormal DNA using analytical flow cytometry and digital image analysis. Other factors such as membrane associated urokinase, increased DNA synthesis rates and certain receptors can be used in the method for detection of potentially invasive tumors.

Novel actions of tissue-type plasminogen activator in chronic kidney disease: a paradigm shift

PubMed Central

Hu, Kebin; Mars, Wendy M.; Liu, Youhua

2009-01-01

Tissue-type plasminogen activator (tPA) is traditionally viewed as a simple serine protease whose main function is to convert plasminogen into biologically active plasmin. As a protease, tPA plays a crucial role in regulating blood fibrinolysis, in maintaining the homeostasis of extracellular matrix (ECM) and in modulating the post-translational activation of growth factors. However, emerging evidence indicates that tPA may also function as a cytokine that transmits its signal across the cell membrane, initiates a diverse array of intracellular signaling, and dictates gene expression in the nuclei. Structurally, tPA is a kringle-containing protein that shares significant similarity to other classic cytokines such as hepatocyte growth factor (HGF) and macrophage-stimulating protein (MSP). Although there is no dedicated receptor, tPA binds to the cell membrane low density lipoprotein (LDL) receptor-related protein-1 (LRP-1), triggers LRP-1 tyrosine phosphorylation, and activates various intracellular signaling. As a cytokine, tPA plays a pivotal role in the pathogenesis of renal interstitial fibrosis through diverse mechanisms. It induces matrix matelloproteinase-9 (MMP-9) gene expression in renal interstitial fibroblasts, which causes the destruction of the tubular basement membrane (TBM), thereby facilitating tubular epithelial to mesenchymal transition (EMT). tPA also potentiates myofibroblast activation from quiescent interstitial fibroblasts through LRP-1-mediated recruitment of β1 integrin signaling. Furthermore, tPA acts as a survival factor that protects renal interstitial fibroblasts/myofibroblasts from apoptosis, thereby resulting in an expansion of myofibroblast populations in diseased kidney. Together, a growing body of evidence has implicated tPA as a fibrogenic cytokine that promotes the progression of kidney diseases. These new findings have radically changed our conception of tPA in renal fibrogenesis and represent a paradigm shift towards uncovering its cytokine function. A better understanding of renal tPA biology will ultimately translate into more rational therapeutic remedies for patients with chronic kidney fibrosis. PMID:18508579

[A 50-year history of new drugs in Japan-the development and trends of hemostatics and antithrombotic drugs].

PubMed

Ozawa, Hikaru; Abiko, Yasushi; Akimoto, Takeshi

2003-01-01

The developments and trends of hemostatic and antithrombotic drugs in Japan were investigated chronologically for the last 50 years after the 2nd World War. 1. Hemostatic drugs are classified into three groups ; capillary stabilizers, blood coagulants and antifibrinolytics. l) As to capillary stabilizers, flavonoid (rutin, 1949), adrenochrome derivative (carbazochrome, 1954) and conjugated estrogen (Premarin, 1964) were introduced therapeutically. Especially, the soluble types of adrenochrome compounds (Adona 1956, S-Adchnon, 1962) were devised and used widely in Japan. 2) Drugs concerning blood coagulation, thrombin, introduced in 1953, and hemocoagulase, a snake venom introduced in 1966, were used clinically. V.K. groups producing various coagulation factors were introduced as V.K1 (Phytonadione, 1962) and V.K2 (rnenatetrenone,1972), and they were admitted in "The Japanese Pharmacopoeia"editions 8 and 14, respectively). 3) Regarding antifibrinolytic drugs, Japanese researchers have made remarkable contributions. e-Aminocapronic acid (Ipsilon, 1962) and tranexamic acid (Transamin, 1965) were developed and used for various abnormal bleedings or hemorrhage associated with plasmin over-activation. tranexamic acid also proved to suppress inflammations of the throat such as tonsillitis, pharyngitis or laryngitis. 2. Antithrombotic drugs are also divided into three groups; anticoagulants, antiplatelet drugs and fibrinolytics.1) The anticoagulants used therapeutically by injection are heparins (Na-salt, 1951; Ca-salt, 1962) and low-molecular-weight heparins such as dalteparin (1992), parnaparin (1994) and reviparin (1999). The low molecule compounds are superior to the original heparins in reducing the risk of bleeding. As oral anticoagulants, coumarin derivatives, dicumarol (1950), ethylbiscoumacetate (1954), phenylindandione (1956) and warfarin (1962) are known. Warfarin potassium is the main drug for oral therapy of thromboembolism lately. Gabexate mesilate (1989) and nafamostat mesilate (1989) were developed in Japan and used for DIC and acute pancreatitis to inhibit protease enzymes. Argatroban is a unique antithrombin product developed by Japanese researchers in 1990, and is used for vascular or cerebral thrombosis. After noticing in 1968 that aspirin inhibits platelet aggregation and prevents myocardial infraction, projects for developing antiplatelet drugs were initiated worldwide. Ticlopidine, originally developed in France, was introduced in 1981 and prevailed widely in Japan for reducing the risk of thrombotic stroke. Aspirin itself was recognized by the FDA (USA) as an antithrombotic drug in 1988, and was also approved by Japanese authorities in 2000. PGE1 clathrate compounds have also been developed as antiplatelet drugs; alprostadil alfadex for injection (1979), and limaprost alfadex for oral use (1988). The PGI2 product, beraprost sodium, for oral use followed them in 1992. Other antiplatelet drugs with unique mechanisms explored in Japan: Ozagrel (1988), which inhibits TXA2 synthetase, cilostazol (1988), which inhibits cAMP phosphodiesterase, and sarpogrelate (1993), which blocks 5HT in platelets, are the notable drugs in this field. Ethyl icosapentate, from fish oil, is available for antiplatelet therapy. Concerning the fibrinolytic system, plasminogen activators are useful for thromboembolism. The streptokinase from bacterial origin developed in the USA and Europe was not introduced, and urokinase (1965) was the first plasminogen activator developed in Japan. Then tissue plasminogen activators (t-PA) tisokinase (cell culture, 1991), alteplase (genetical recombination, 1991), nateplase (genetical recombination, 1996), monteplase (1998) and pamiteplase (1998) were developed and approved for acute myocardial infarction. Nasaruplase (prourokinase, cell culture,1991) was also approved for the same indication. While the development of the hemostatic drugs ceased in the 1960s, avid project studies for antithrombotic drugs including fibrinolytics began in the 1980s and are progressing now towards new molecular targets. This may be due to the increasing tendency of cardiovascular thromboembolic diathesis in Japan. (The figures in parentheses are the years approved by the Japanese Ministry of Health, Labor and Welfare.)

Ganodermanontriol (GDNT) exerts its effect on growth and invasiveness of breast cancer cells through the down-regulation of CDC20 and uPA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Jiahua; Jedinak, Andrej; Sliva, Daniel, E-mail: dsliva@iuhealth.org

2011-11-18

Highlights: Black-Right-Pointing-Pointer Ganodermanontriol (GDNT), a Ganoderma mushroom alcohol, inhibits growth of breast cancer cells. Black-Right-Pointing-Pointer CDC20 is over-expressed in tumors but not in the tumor surrounding tissue in breast cancer patients. Black-Right-Pointing-Pointer GDNT inhibits expression of CDC20 in breast cancer cells. Black-Right-Pointing-Pointer GDNT inhibits cell adhesion, cell migration and cell invasion of breast cancer cells. Black-Right-Pointing-Pointer GDNT inhibits secretion of uPA and down-regulates expression of uPAR in breast cancer cells. -- Abstract: Ganoderma lucidum is a medicinal mushroom that has been recognized by Traditional Chinese Medicine (TCM). Although some of the direct anticancer activities are attributed to the presence ofmore » triterpenes-ganoderic and lucidenic acids-the activity of other compounds remains elusive. Here we show that ganodermanontriol (GDNT), a Ganoderma alcohol, specifically suppressed proliferation (anchorage-dependent growth) and colony formation (anchorage-independent growth) of highly invasive human breast cancer cells MDA-MB-231. GDNT suppressed expression of the cell cycle regulatory protein CDC20, which is over-expressed in precancerous and breast cancer cells compared to normal mammary epithelial cells. Moreover, we found that CDC20 is over-expressed in tumors when compared to the tissue surrounding the tumor in specimens from breast cancer patients. GDNT also inhibited invasive behavior (cell adhesion, cell migration, and cell invasion) through the suppression of secretion of urokinase-plasminogen activator (uPA) and inhibited expression of uPA receptor. In conclusion, mushroom GDNT is a natural agent that has potential as a therapy for invasive breast cancers.« less

Possible mechanisms for arsenic-induced proliferative diseases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wetterhahn, K.E.; Dudek, E.J.; Shumilla, J.A.

1996-12-31

Possible mechanisms for cardiovascular diseases and cancers which have been observed on chronic exposure to arsenic have been investigated. We tested the hypothesis that nonlethal levels of arsenic are mitogenic, cause oxidative stress, increase nuclear translocation of trans-acting factors, and increase expression of genes involved in proliferation. Cultured porcine vascular (from aorta) endothelial cells were used as a model cell system to study the effects of arsenic on the target cells for cardiovascular diseases. Treatment of postconfluent cell cultures with nonovertly toxic concentrations of arsenite increased DNA synthesis, similar to the mitogenic response observed with hydrogen peroxide. Within 1 hourmore » of adding noncytotoxic concentrations of arsenite, cellular levels of oxidants increased relative to control levels, indicating that arsenite promotes cellular oxidations. Arsenite treatment increased nuclear translocation of NF-{kappa}B, an oxidative stress-responsive transcription factor, in a manner similar to that observed with hydrogen peroxide. Pretreatment of intact cells with the antioxidants N-acetylcysteine and dimethylfumarate prevented the arsenite-induced increases in cellular oxidant formation and NF-KB translocation. Arsenite had little or no effect on binding of NF-KB to its DNA recognition sequence in vitro, indicating that it is unlikely that arsenite directly affects NF-KB. The steady-state mRNA levels of intracellular adhesion molecule and urokinase-like plasminogen activator, genes associated with the active endothelial phenotype in arteriosclerosis and cancer metastasis, were increased by nontoxic concentrations of arsenite. These data suggest that arsenite promotes proliferative diseases like heart disease and cancer by activating oxidant-sensitive endothelial cell signaling and gene expression. It is possible that antioxidant therapy would be useful in preventing arsenic-induced cardiovascular disease and cancer.« less

The anti-metastatic effects of the phytoestrogen arctigenin on human breast cancer cell lines regardless of the status of ER expression.

PubMed

Maxwell, Thressi; Chun, So-Young; Lee, Kyu-Shik; Kim, Soyoung; Nam, Kyung-Soo

2017-02-01

Arctigenin is a plant lignan extracted from Arctium lappa that has been shown to have estrogenic properties. In spite of the health benefits of phytoestrogens reducing the risk of osteoporosis, heart disease, and menopausal symptoms, its benefits against the risk of breast cancer have not been fully elucidated. Thus, we investigated the effects of arctigenin on metastasis of breast cancer using both estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cell lines to see if the effects are dependent on the status of ER expression. In ER-positive MCF-7 cells, arctigenin efficiently inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell migration and invasion. The activity of crucial metastatic protease matrix metalloprotease (MMP)-9 in gelatin zymography was also efficiently decreased by arctigenin, as well as its mRNA expression. Notably, arctigenin exhibited similar anti-metastatic effects even in ER-negative MDA-MB-231 cells, suggesting that the anti-metastatic effects of arctigenin were not exerted via the ER. The upstream signaling pathways involved in the regulation of MMP-9 and urokinase plasminogen activator (uPA) were analyzed using western blotting. The activation of Akt, NF-κB and MAPK (ERK 1/2 and JNK 1/2) was found to be inhibited. Taken together, these data suggest that arctigenin confers anti-metastatic effects by inhibiting MMP-9 and uPA via the Akt, NF-κB and MAPK signaling pathways on breast cancer, regardless of ER expression. Therefore, we propose that the intake of arctigenin could be an effective supplement for breast cancer patients.

Telemedicine Can Replace the Neurologist on a Mobile Stroke Unit.

PubMed

Wu, Tzu-Ching; Parker, Stephanie A; Jagolino, Amanda; Yamal, Jose-Miguel; Bowry, Ritvij; Thomas, Abraham; Yu, Amy; Grotta, James C

2017-02-01

The BEST-MSU study (Benefits of Stroke Treatment Delivered Using a Mobile Stroke Unit) is a comparative effectiveness trial in patients randomized to mobile stroke unit or standard management. A substudy tested interrater agreement for tissue-type plasminogen activator eligibility between a telemedicine vascular neurologist and onboard vascular neurologist. On scene, both the telemedicine vascular neurologist and onboard vascular neurologist independently evaluated the patient, documenting their tissue-type plasminogen activator treatment decision, National Institutes of Health Stroke Scale score, and computed tomographic interpretation. Agreement was determined using Cohen κ statistic. Telemedicine-related technical failures that impeded remote assessment were recorded. Simultaneous and independent telemedicine vascular neurologist and onboard vascular neurologist assessment was attempted in 174 patients. In 4 patients (2%), the telemedicine vascular neurologist could not make a decision because of technical problems. The telemedicine vascular neurologist agreed with the onboard vascular neurologist on 88% of evaluations (κ=0.73). Remote telemedicine vascular neurologist assessment is reliable and accurate, supporting either telemedicine vascular neurologist or onboard vascular neurologist assessment on our mobile stroke unit. URL: http://www.clinicaltrials.gov. Unique identifier: NCT02190500. © 2017 American Heart Association, Inc.

Plasminogen activator inhibitor-1 4G/5G polymorphism is associated with type 2 diabetes risk

PubMed Central

Zhao, Luqian; Huang, Ping

2013-01-01

A number of studies were performed to assess the association between plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism and susceptibility to type 2 diabetes (T2DM). However, the results were inconsistent and inconclusive. In the present study, the possible association was investigated by a meta-analysis. Eligible articles were identified for the period up to June 2013. Pooled odds ratios (OR) with 95% confidence intervals (CI) were appropriately derived from random-effects models or fixed-effects models. Fourteen case-control studies with a total of 2487 cases and 3538 controls were eligible. In recessive model, PAI-1 4G/5G polymorphism was associated with T2DM risk (OR = 1.23; 95% CI 1.07-1.41; P = 0.004). In the subgroup analysis by ethnicity, a significant association was found among Asians (OR = 1.27; 95% CI 1.08-1.51; P = 0.005). This meta-analysis suggested that PAI-1 4G/5G polymorphism may be associated with T2DM development. PMID:24040470

Unusual cause of aborted sudden cardiac death in a teen athlete: homozygosity for the 4G allele of the plasminogen activase inhibitor type 1 gene.

PubMed

Phillips, Susie B; Batlivala, Sarosh; Knudson, Jarrod D

2015-10-01

Common aetiologies of sudden cardiac death in children include coronary anomalies, channelopathies, and cardiomyopathies. Less frequently, hypercoagulable states cause sudden arrest. We report an unusual case of aborted sudden cardiac death in a teenager, ultimately found to have homozygosity for the 4G allele of the plasminogen activase inhibitor type 1 gene.

Plasmin-dependent proteolysis of Tissue Factor Pathway Inhibitor in a mouse model of endotoxemia

PubMed Central

Lupu, Cristina; Herlea, Oana; Tang, Haiwang; Lijnen, Roger H.; Lupu, Florea

2012-01-01

Summary Background Development of a procoagulant state in sepsis, due to aberrant expression of tissue factor (TF) and sharp decrease of its major inhibitor tissue factor pathway inhibitor (TFPI), could lead to microthrombotic organ failure. The mechanism for the decline of TFPI activity in the lung could involve plasmin-mediated cleavage of the inhibitor. Objective To investigate the effect of plasmin generation on lung-associated TFPI activity, in normal conditions and during infusion of endotoxin (LPS) in mice. Methods Plasmin generation and TFPI activity were assayed in the lungs of mice deficient of tissue-type plasminogen activator (t-PA) or plasminogen (Plg), at 2-hrs after LPS or saline injection. Results The sharp loss of lung-associated TFPI activity at 2-hrs post LPS paralleled the abrupt increase of plasmin generation. TFPI activity was significantly retained in both t-PA-/- and Plg-/- mice, which are unable to generate plasmin. Conclusion The increased plasmin generation during the early stages of sepsis could cleave/inactivate TFPI and thus lead to thrombotic complications. PMID:23106863

Adolescents with Classical Polycystic Ovary Syndrome Have Alterations in the Surrogate Markers of Cardiovascular Disease but Not in the Endothelial Function. The Possible Benefits of Metformin.

PubMed

Fruzzetti, Franca; Ghiadoni, Lorenzo; Virdis, Agostino; De Negri, Ferdinando; Perini, Daria; Bucci, Fiorella; Giannarelli, Chiara; Gadducci, Angiolo; Taddei, Stefano

2016-10-01

To study whether adolescents with the classical form of polycystic ovary syndrome (PCOS) have alterations in metabolic and vascular structure and function. The effect of metformin was evaluated. Controlled study. University outpatient clinic. Eighteen nonobese adolescents with PCOS were enrolled. Seventeen healthy age-matched adolescents were recruited as control subjects. The metabolic profile and the endothelial structure and function were evaluated. Hormonal and lipid profile, blood pressure (BP) measurement, fasting glucose and insulin levels, C-reactive protein (CRP), homocysteine, tissue-type plasminogen activator, plasminogen activator inhibitor-1 (PAI-1), and plasmin-antiplasmin complexes (PAP) were measured. Flow mediated dilation (FMD), central pulse wave velocity (PWV), radial artery pulse wave, and common carotid intima-media thickness (IMT) were also assessed. Girls with PCOS were also studied 6 months after treatment with metformin (850 mg twice per day). Adolescents with PCOS were insulin resistant and/or hyperinsulinemic and they had higher BP values and levels of CRP and PAI-1 than the control subjects. The levels of tissue-type plasminogen activator and PAP were similar in both groups. FMD, PWV, and IMT were also similar. Metformin significantly (P < .05) reduced insulin, BP, CRP, and PAI-1 levels. The PAP levels significantly (P < .05) increased. Radial artery pulse wave was significantly reduced after metformin treatment. No modifications in FMD, PWV, and IMT were observed. Adolescents with classical PCOS have alterations in some surrogate markers of cardiovascular risk and they are ameliorated by metformin. No deterioration of vascular structure and function has been detected, probably because of the short duration of exposure to the disease. Copyright © 2016 North American Society for Pediatric and Adolescent Gynecology. Published by Elsevier Inc. All rights reserved.

Sickle Mice Are Sensitive to Hypoxia/Ischemia-Induced Stroke but Respond to Tissue-Type Plasminogen Activator Treatment.

PubMed

Sun, Yu-Yo; Lee, Jolly; Huang, Henry; Wagner, Mary B; Joiner, Clinton H; Archer, David R; Kuan, Chia-Yi

2017-12-01

The effects of lytic stroke therapy in patients with sickle cell anemia are unknown, although a recent study suggested that coexistent sickle cell anemia does not increase the risk of cerebral hemorrhage. This finding calls for systemic analysis of the effects of thrombolytic stroke therapy, first in humanized sickle mice, and then in patients. There is also a need for additional predictive markers of sickle cell anemia-associated vasculopathy. We used Doppler ultrasound to examine the carotid artery of Townes sickle mice tested their responses to repetitive mild hypoxia-ischemia- and transient hypoxia-ischemia-induced stroke at 3 or 6 months of age, respectively. We also examined the effects of tPA (tissue-type plasminogen activator) treatment in transient hypoxia-ischemia-injured sickle mice. Three-month-old sickle cell (SS) mice showed elevated resistive index in the carotid artery and higher sensitivity to repetitive mild hypoxia-ischemia-induced cerebral infarct. Six-month-old SS mice showed greater resistive index and increased flow velocity without obstructive vasculopathy in the carotid artery. Instead, the cerebral vascular wall in SS mice showed ectopic expression of PAI-1 (plasminogen activator inhibitor-1) and P-selectin, suggesting a proadhesive and prothrombotic propensity. Indeed, SS mice showed enhanced leukocyte and platelet adherence to the cerebral vascular wall, broader fibrin deposition, and higher mortality after transient hypoxia-ischemia. Yet, post-transient hypoxia-ischemia treatment with tPA reduced thrombosis and mortality in SS mice. Sickle mice are sensitive to hypoxia/ischemia-induced cerebral infarct but benefit from thrombolytic treatment. An increased resistive index in carotid arteries may be an early marker of sickle cell vasculopathy. © 2017 American Heart Association, Inc.

The metabolic syndrome is associated with a higher resistance to intravenous thrombolysis for acute ischemic stroke in women than in men.

PubMed

Arenillas, Juan F; Sandoval, Patricio; Pérez de la Ossa, Natalia; Millán, Mónica; Guerrero, Cristina; Escudero, Domingo; Dorado, Laura; López-Cancio, Elena; Castillo, José; Dávalos, Antoni

2009-02-01

The metabolic syndrome (MetS) might confer a higher resistance to intravenous thrombolysis in acute middle cerebral artery (MCA) ischemic stroke. MetS increases the risk of stroke in women to a greater extent than in men. We aimed to investigate whether there might be sex differences in the impact of MetS on the response to intravenous thrombolysis for acute MCA ischemic stroke. We prospectively studied consecutive ischemic stroke patients, treated with intravenous tissue-type plasminogen activator according to SITS-MOST criteria, with an MCA occlusion on prebolus transcranial Doppler examination. Resistance to thrombolysis was defined as the absence of complete MCA recanalization 24 hours after tissue-type plasminogen activator infusion by transcranial Doppler criteria. MetS was diagnosed according to the criteria established by the American Heart Association/National Heart, Lung, and Blood Institute 2005 statement. A total of 125 patients (75 men, 50 women; mean age, 67.6+/-11 years) were included. MetS was diagnosed in 76 (61%) patients. Resistance to clot lysis at 24 hours was observed in 53 (42%) patients. Two multivariate-adjusted, logistic-regression models identified that MetS was associated with a higher resistance to tissue-type plasminogen activator, independently of other significant baseline variables (odds ratio=9.8; 95% CI, 3.5 to 27.8; P=0.0001) and of the individual components of the MetS. The MetS was associated with a significantly higher odds of resistance to thrombolysis in women (odds ratio=17.5; 95% CI, 1.9 to 163.1) than in men (odds ratio=5.1; 95% CI, 1.6 to 15.6; P for interaction=0.0004). The effect of MetS on the resistance to intravenous thrombolysis for acute MCA ischemic stroke appears to be more pronounced in women than in men.

The plasminogen activator system modulates sympathetic nerve function.

PubMed

Schaefer, Ulrich; Machida, Takuji; Vorlova, Sandra; Strickland, Sidney; Levi, Roberto

2006-09-04

Sympathetic neurons synthesize and release tissue plasminogen activator (t-PA). We investigated whether t-PA modulates sympathetic activity. t-PA inhibition markedly reduced contraction of the guinea pig vas deferens to electrical field stimulation (EFS) and norepinephrine (NE) exocytosis from cardiac synaptosomes. Recombinant t-PA (rt-PA) induced exocytotic and carrier-mediated NE release from cardiac synaptosomes and cultured neuroblastoma cells; this was a plasmin-independent effect but was potentiated by a fibrinogen cleavage product. Notably, hearts from t-PA-null mice released much less NE upon EFS than their wild-type (WT) controls (i.e., a 76.5% decrease; P<0.01), whereas hearts from plasminogen activator inhibitor-1 (PAI-1)-null mice released much more NE (i.e., a 275% increase; P<0.05). Furthermore, vasa deferentia from t-PA-null mice were hyporesponsive to EFS (P<0.0001) but were normalized by the addition of rt-PA. In contrast, vasa from PAI-1-null mice were much more responsive (P<0.05). Coronary NE overflow from hearts subjected to ischemia/reperfusion was much smaller in t-PA-null than in WT control mice (P<0.01). Furthermore, reperfusion arrhythmias were significantly reduced (P<0.05) in t-PA-null hearts. Thus, t-PA enhances NE release from sympathetic nerves and contributes to cardiac arrhythmias in ischemia/reperfusion. Because the risk of arrhythmias and sudden cardiac death is increased in hyperadrenergic conditions, targeting the NE-releasing effect of t-PA may have valuable therapeutic potential.

[Construction and expression of a fusion protein made of tissue-type plasminogen activator and hirudin in Pichia pastoris].

PubMed

Yu, Ai-Ping; Shi, Bing-Xing; Dong, Chun-Na; Jiang, Zhong-Hua; Wu, Zu-Ze

2005-07-01

To combine the fibrinolytic with anticoagulant activities for therapy of thrombotic deseases, a fusion protein made of tissue-type plasminogen activator (t-PA) and hirudin was constructed and expressed in chia pastoris. To improve thrombolytic properties of t-PA and reduce bleeding side effect of hirudin, FXa-recognition sequence was introduced between t-PA and hirudin molecules.The anticoagulant activity of hirudin can be target-released through cleavage of FXa at thrombus site. t-PA gene and hirudin gene with FXa-recognition sequence at its 5'-terminal were obtained by RT-PCR and PCR respectively. The fusion protein gene was cloned into plasmid pIC9K and electroporated into the genome of Pichia pastoris GS115. The expression of fusion protein was induced by methanol in shaking flask and secreted into the culture medium. Two forms of the fusion protein, single-chain and double-chain linked by a disulfide bond (due to the cleveage of t-PA at Arg275-Ile276), were obtained. The intact fusion protein retained the fibrinolytic activity but lacked any anticoagulant activity. After cleavage by FXa, the fusion protein liberated intact free hirudin to exert its anticoagulant activity. So, the fusion protein is a bifunctional molecule having good prospect to develop into a new targeted therapeutic agent with reduced bleeding side effect for thrombotic diseases.

Recombinant tissue plasminogen activator as a novel treatment option for infective endocarditis: a retrospective clinical study in 32 children.

PubMed

Levitas, Aviva; Krymko, Hanna; Richardson, Justin; Zalzstein, Eli; Ioffe, Viktoriya

2016-01-01

Infective endocarditis is a life-threatening infectious syndrome, with high morbidity and mortality. Current treatments for infective endocarditis include intravenous antibiotics, surgery, and involve a lengthy hospital stay. We hypothesised that adjunctive recombinant tissue plasminogen activator treatment for infective endocarditis may facilitate faster resolution of vegetations and clearance of positive blood cultures, and therefore decrease morbidity and mortality. This retrospective study included follow-up of patients, from 1997 through 2014, including clinical presentation, causative organism, length of treatment, morbidity, and mortality. We identified 32 patients, all of whom were diagnosed with endocarditis and were treated by recombinant tissue plasminogen activator. Among all, 27 patients (93%) had positive blood cultures, with the most frequent organisms being Staphylococcus epidermis (nine patients), Staphylococcus aureus (six patients), and Candida (nine patients). Upon treatment, in 31 patients (97%), resolution of vegetations and clearance of blood cultures occurred within hours to few days. Out of 32 patients, one patient (3%) died and three patients (9%) suffered embolic or haemorrhagic events, possibly related to the recombinant tissue plasminogen activator. None of the patients required surgical intervention to assist vegetation resolution. In conclusion, it appears that recombinant tissue plasminogen activator may become an adjunctive treatment for infective endocarditis and may decrease morbidity as compared with current guidelines. Prospective multi-centre studies are required to validate our findings.

Role of p53–fibrinolytic system cross-talk in the regulation of quartz-induced lung injury

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhandary, Yashodhar P.; Shetty, Shwetha K.; Marudamuthu, Amarnath S.

2015-03-01

Silica is the major component of airborne dust generated by wind, manufacturing and/or demolition. Chronic occupational inhalation of silica dust containing crystalline quartz is by far the predominant form of silicosis in humans. Silicosis is a progressive lung disease that typically arises after a very long latency and is a major occupational concern with no known effective treatment. The mechanism of silicosis is not clearly understood. However, silicosis is associated with increased cell death, expression of redox enzymes and pro-fibrotic cytokines and chemokines. Since alveolar epithelial cell (AEC) death and disruption of alveolar fibrinolysis is often associated with both acutemore » and chronic lung injuries, we explored whether p53-mediated changes in the urokinase-type plasminogen activator (uPA) system contributes to silica-induced lung injury. We further sought to determine whether caveolin-1 scaffolding domain peptide (CSP), which inhibits p53 expression, mitigates lung injury associated with exposure to silica. Lung tissues and AECs isolated from wild-type (WT) mice exposed to silica exhibit increased apoptosis, p53 and PAI-1, and suppression of uPA expression. Treatment of WT mice with CSP inhibits PAI-1, restores uPA expression and prevents AEC apoptosis by suppressing p53, which is otherwise induced in mice exposed to silica. The process involves CSP-mediated inhibition of serine-15 phosphorylation of p53 by inhibition of protein phosphatase 2A-C (PP2A-C) interaction with silica-induced caveolin-1 in AECs. These observations suggest that changes in the p53–uPA fibrinolytic system cross-talk contribute to lung injury caused by inhalation of silica dust containing crystalline quartz and is protected by CSP by targeting this pathway. - Highlights: • Chronic exposure to quartz dusts is a major cause of lung injury and silicosis. • The survival of patients with silicosis is bleak due to lack of effective treatments. • This study defines a new role of p53–uPA cross-talk in quartz-induced lung injury. • Targeting the p53–uPA system inhibits ATII cell/lung injury due to quartz exposure.« less

Inhibition of breast cancer metastasis by co-transfection of miR-31/193b-mimics

PubMed Central

Hashemi, Zahra Sadat; Moghadam, Mehdi Forouzandeh; Farokhimanesh, Samila; Rajabibazl, Masoumeh; Sadroddiny, Esmaeil

2018-01-01

Objective(s): Various studies have been conducted to reduce the metastatic behavior of cancerous cells. In this regard, ectopic expression of anti-metastatic microRNAs by miR-mimic and miR-restoration-based therapies could bring new insights to the field. In the present study, the consequences of co-transfecting breast cancer cell lines with miR-193b and miR-31 were investigated via invasion and migration assays. Materials and Methods: Double stranded oligonucleotide of mature miR-193b-3p and miR-31-5p were cloned into pcDNA 6.2gw/EmGFP plasmid. The resulting plasmids were used for transfection. Real time-PCR was performed to assess the expression of miR-193b and miR-31 as well as Ras homolog gene family member A (RhoA) and urokinase-type plasminogen activator (uPA) as miR targets. Scratch, Transwell migration and Matrigel invasion assays were carried out to assess the extent of migration and invasion of cell lines. Results: The most significant increase in expression of miRs belonged to the single transfection of mimic-miRs in MDA-MB231. Although the co-transfection was not as successful as single transfection in miR expression, it was significantly more effective in inhibition of the cells invasive potential. Conclusion: Although the miR-restoration therapy based on co-transfection of two miRs could be less effective in expression of each miRNA, the resulting decrease in metastatic behavior of the cells is more significant due to collective effect of co-transfection to decrease target gene expression. Our results revealed that employing this sort of combinatorial strategies could lead to more efficient reduction in metastatic behavior. It seems that using this strategy would bring about more successful therapeutic outcomes. PMID:29796229

The Significance of Notch1 Compared with Notch3 in High Metastasis and Poor Overall Survival in Hepatocellular Carcinoma

PubMed Central

Li, Qingjun; Sun, Wei; Zhang, Yong; Wang, Desheng; Dou, Kefeng

2013-01-01

Background The prognosis for patients with hepatocellular carcinoma (HCC) is poor, and the mechanisms underlying the development of HCC remain unclear. Notch1 and Notch3 may be involved in malignant transformation, although their roles remain unknown. Materials and Methods HCC tissues were stained with anti-Notch1 or -Notch3 antibody. The migration and invasion capacities of the cells were measured with transwell cell culture chambers. RT-PCR was used to measure the expression of Notch1 and Notch3 mRNA. Additionally, western blot analysis was used to assess the protein expression of Notch1, Notch3, CD44v6, E-cadherin, matrix metalloproteinase-2 (MMP-2), MMP-9, and urokinase-type plasminogen activator (uPA). RNA interference was used to down-regulate the expression of Notch1 and Notch3. Cell viability was assessed using MTT. Results Based on immunohistochemistry, high Notch1 expression was correlated with tumor size, tumor grade, metastasis, venous invasion and AJCC TNM stage. High Notch3 expression was only strongly correlated with metastasis, venous invasion and satellite lesions. Kaplan-Meier curves demonstrated that patients with high Notch1 or Notch3 expression were at a significantly increased risk for shortened survival time. In vitro, the down-regulation of Notch1 decreased the migration and invasion capacities of HCC cells by regulating CD44v6, E-cadherin, MMP-2, MMP-9, and uPA via the COX-2 and ERK1/2 pathways. Down-regulation of Notch3 only decreased the invasion capacity of HCC cells by regulating MMP-2 and MMP-9 via the ERK1/2 pathway. Conclusions Based on the migration and invasion of HCC, we hypothesize that targeting Notch1 may be more useful than Notch3 for designing novel preventive and therapeutic strategies for HCC in the near future. PMID:23468978

Isothiocyanate-Functionalized Bifunctional Chelates and fac-[M(I)(CO)3](+) (M = Re, (99m)Tc) Complexes for Targeting uPAR in Prostate Cancer.

PubMed

Kasten, Benjamin B; Ma, Xiaowei; Cheng, Kai; Bu, Lihong; Slocumb, Winston S; Hayes, Thomas R; Trabue, Steven; Cheng, Zhen; Benny, Paul D

2016-01-20

Developing new strategies to rapidly incorporate the fac-[M(I)(CO)3](+) (M = Re, (99m)Tc) core into biological targeting vectors in radiopharmaceuticals continues to expand as molecules become more complex and as efforts to minimize nonspecific binding increase. This work examines a novel isothiocyanate-functionalized bifunctional chelate based on 2,2'-dipicolylamine (DPA) specifically designed for complexing the fac-[M(I)(CO)3](+) core. Two strategies (postlabeling and prelabeling) were explored using the isothiocyanate-functionalized DPA to determine the effectiveness of assembly on the overall yield and purity of the complex with amine containing biomolecules. A model amino acid (lysine) examined (1) amine conjugation of isothiocyanate-functionalized DPA followed by complexation with fac-[M(I)(CO)3](+) (postlabeling) and (2) complexation of fac-[M(I)(CO)3](+) with isothiocyanate-functionalized DPA followed by amine conjugation (prelabeling). Conducted with stable Re and radioactive (99m)Tc analogs, both strategies formed the product in good to excellent yields under macroscopic and radiotracer concentrations. A synthetic peptide (AE105) which targets an emerging biomarker in CaP prognosis, urokinase-type plasminogen activator receptor (uPAR), was also explored using the isothiocyanate-functionalized DPA strategy. In vitro PC-3 (uPAR+) cell uptake assays with the (99m)Tc-labeled peptide (8a) showed 4.2 ± 0.5% uptake at 4 h. In a murine model bearing PC-3 tumor xenografts, in vivo biodistribution of 8a led to favorable tumor uptake (3.7 ± 0.7% ID/g) at 4 h p.i. with relatively low accumulation (<2% ID/g) in normal organs not associated with normal peptide excretion. These results illustrate the promise of the isothiocyanate-functionalized approach for labeling amine containing biological targeting vectors with fac-[M(I)(CO)3](+).

A comparative analysis of novel cardiovascular biomarkers in patients with chronic heart failure.

PubMed

Lichtenauer, Michael; Jirak, Peter; Wernly, Bernhard; Paar, Vera; Rohm, Ilonka; Jung, Christian; Schernthaner, Christiana; Kraus, Johannes; Motloch, Lukas J; Yilmaz, Atilla; Hoppe, Uta C; Christian Schulze, P; Kretzschmar, Daniel; Pistulli, Rudin

2017-10-01

Heart failure (HF) with reduced ejection fraction remains a major therapeutic challenge. The aim of this study was to investigate the role of novel cardiovascular biomarkers, i.e. soluble suppression of tumorigenicity (sST2), growth-differentiation factor-15 (GDF-15), soluble urokinase plasminogen activator receptor (suPAR) and heart-type fatty acid binding protein (H-FABP) in patients with ischaemic (ICM) or dilative cardiomyopathy (DCM). A total of 200 patients were enrolled in this study: 65 were diagnosed with DCM and 59 patients suffering from ICM were included. 76 patients without coronary artery disease or signs of heart failure were included as controls. Plasma samples of all patients were analyzed by use of ELISA. Levels of sST2, suPAR and H-FABP were significantly higher in ICM and DCM patients compared to the control group (p<0.0001). However, there were no significant differences between ICM and DCM in biomarker levels. Ejection fraction correlated inversely with cardiac biomarkers (sST2 p<0.0001, GDF-15 p=0.0394, suPAR p=0.0029, H-FABP p<0.0001). Similarly, CRP levels also showed a positive correlation with cardiac biomarkers. Renal insufficiency (p<0.0001) and diabetes (sST2 p=0.0021, GDF-15 p=0.0055, suPAR p=0.0339, H-FABP p=0.0010) were significantly associated with a rise in cardiac biomarkers. Novel cardiovascular biomarkers such as ST2, GDF-15, uPAR and H-FABP could offer a great potential for more precise diagnostic in ICM and DCM patients. H-FABP was the most promising marker in our study, followed by sST2, uPAR and GDF-15. Additional prospective studies will be necessary to further evaluate the potential clinical benefits in routine treatment of HF. Copyright © 2017 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.

Overexpression of EMMPRIN Isoform 2 Is Associated with Head and Neck Cancer Metastasis

PubMed Central

Guo, Weijie; Wang, Lili; Li, Haigang; Zhang, Tianyu; Liu, Xiaojia; Xu, Qin; Li, Jinsong; Guo, Zhongmin

2014-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN), a plasma membrane protein of the immunoglobulin (Ig) superfamily, has been reported to promote cancer cell invasion and metastasis in several human malignancies. However, the roles of the different EMMPRIN isoforms and their associated mechanisms in head and neck cancer progression remain unknown. Using quantitative real-time PCR, we found that EMMPRIN isoform 2 (EMMPRIN-2) was the only isoform that was overexpressed in both head and neck cancer tissues and cell lines and that it was associated with head and neck cancer metastasis. To determine the effects of EMMPRIN-2 on head and neck cancer progression, we transfected head and neck cancer cells with an EMMPRIN-2 expression vector and EMMPRIN-2 siRNA to exogenously modulate EMMPRIN-2 expression and examined the functional importance of EMMPRIN-2 in head and neck cancer invasion and metastasis. We found that EMMPRIN-2 promoted head and neck cancer cell invasion, migration, and adhesion in vitro and increased lung metastasis in vivo. Mechanistic studies revealed that EMMPRIN-2 overexpression promoted the secretion of extracellular signaling molecules, including matrix metalloproteinases-2(MMP-2), urokinase-type plasminogen activator(uPA) and Cathepsin B, in head and neck cancer cells. While MMP-2 and uPA have been demonstrated to be important mediators of EMMPRIN signaling, the role of Cathepsin B in EMMPRIN-mediated molecular cascades and tumorigenesis has not been established. We found that EMMPRIN-2 overexpression and Cathepsin B down-regulation significantly inhibited the invasion, migration and adhesion of Tca8133 cells, suggesting that Cathepsin B is required for EMMPRIN-2 enhanced cell migration and invasion in head and neck cancer. The results of our study demonstrate the important role of EMMPRIN-2 in head and neck cancer progression for the first time and reveal that increased extracellular secretion of Cathepsin B may be a novel mechanism underlying EMMPRIN-2 enhanced tumor progression in head and neck cancer. PMID:24705283

Overexpression of EMMPRIN isoform 2 is associated with head and neck cancer metastasis.

PubMed

Huang, Zhiquan; Tan, Ning; Guo, Weijie; Wang, Lili; Li, Haigang; Zhang, Tianyu; Liu, Xiaojia; Xu, Qin; Li, Jinsong; Guo, Zhongmin

2014-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN), a plasma membrane protein of the immunoglobulin (Ig) superfamily, has been reported to promote cancer cell invasion and metastasis in several human malignancies. However, the roles of the different EMMPRIN isoforms and their associated mechanisms in head and neck cancer progression remain unknown. Using quantitative real-time PCR, we found that EMMPRIN isoform 2 (EMMPRIN-2) was the only isoform that was overexpressed in both head and neck cancer tissues and cell lines and that it was associated with head and neck cancer metastasis. To determine the effects of EMMPRIN-2 on head and neck cancer progression, we transfected head and neck cancer cells with an EMMPRIN-2 expression vector and EMMPRIN-2 siRNA to exogenously modulate EMMPRIN-2 expression and examined the functional importance of EMMPRIN-2 in head and neck cancer invasion and metastasis. We found that EMMPRIN-2 promoted head and neck cancer cell invasion, migration, and adhesion in vitro and increased lung metastasis in vivo. Mechanistic studies revealed that EMMPRIN-2 overexpression promoted the secretion of extracellular signaling molecules, including matrix metalloproteinases-2(MMP-2), urokinase-type plasminogen activator(uPA) and Cathepsin B, in head and neck cancer cells. While MMP-2 and uPA have been demonstrated to be important mediators of EMMPRIN signaling, the role of Cathepsin B in EMMPRIN-mediated molecular cascades and tumorigenesis has not been established. We found that EMMPRIN-2 overexpression and Cathepsin B down-regulation significantly inhibited the invasion, migration and adhesion of Tca8133 cells, suggesting that Cathepsin B is required for EMMPRIN-2 enhanced cell migration and invasion in head and neck cancer. The results of our study demonstrate the important role of EMMPRIN-2 in head and neck cancer progression for the first time and reveal that increased extracellular secretion of Cathepsin B may be a novel mechanism underlying EMMPRIN-2 enhanced tumor progression in head and neck cancer.

A randomized trial of an early measles vaccine at 4½ months of age in Guinea-Bissau: sex-differential immunological effects.

PubMed

Jensen, Kristoffer Jarlov; Søndergaard, Mia; Andersen, Andreas; Sartono, Erliyani; Martins, Cesario; Garly, May-Lill; Eugen-Olsen, Jesper; Ullum, Henrik; Yazdanbakhsh, Maria; Aaby, Peter; Benn, Christine Stabell; Erikstrup, Christian

2014-01-01

After measles vaccine (MV), all-cause mortality is reduced more than can be explained by the prevention of measles, especially in females. We aimed to study the biological mechanisms underlying the observed non-specific and sex-differential effects of MV on mortality. Within a large randomised trial of MV at 4.5 months of age blood samples were obtained before and six weeks after randomisation to early MV or no early MV. We measured concentrations of cytokines and soluble receptors from plasma (interleukin-1 receptor agonist (IL-1Ra), IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, soluble urokinase-type plasminogen activator receptor), and secreted cytokines (interferon-γ, TNF-α, IL-5, IL-10, IL-13, IL-17) after in vitro challenge with innate agonists and recall antigens. We analysed the effect of MV in multiple imputation regression, overall and stratified by sex. The majority of the infants had previously been enrolled in a randomised trial of neonatal vitamin A. Post hoc we explored the potential effect modification by neonatal vitamin A. Overall, MV versus no MV was associated with higher plasma MCP-1 levels, but the effect was only significant among females. Additionally, MV was associated with increased plasma IL-1Ra. MV had significantly positive effects on plasma IL-1Ra and IL-8 levels in females, but not in males. These effects were strongest in vitamin A supplemented infants. Vitamin A shifted the effect of MV in a pro-inflammatory direction. In this explorative study we found indications of sex-differential effects of MV on several of the plasma biomarkers investigated; in particular MV increased levels in females, most strongly in vitamin A recipients. The findings support that sex and micronutrient supplementation should be taken into account when analysing vaccine effects. clinicaltrials.gov number NCT 00168545.

Tob1 induces apoptosis and inhibits proliferation, migration and invasion of gastric cancer cells by activating Smad4 and inhibiting β‑catenin signaling.

PubMed

Kundu, Juthika; Wahab, S M Riajul; Kundu, Joydeb Kumar; Choi, Yoon-La; Erkin, Ozgur Cem; Lee, Hun Seok; Park, Sang Gyu; Shin, Young Kee

2012-09-01

Transducer of ErbB-2.1 (Tob1), a tumor suppressor protein, is inactivated in a variety of cancers including stomach cancer. However, the role of Tob1 in gastric carcinogenesis remains elusive. The present study aimed to investigate whether Tob1 could inhibit gastric cancer progression in vitro, and to elucidate its underlying molecular mechanisms. We found differential expression of Tob1 in human gastric cancer (MKN28, AGS and MKN1) cells. The overexpression of Tob1 induced apoptosis in MKN28 and AGS cells, which was associated with sub-G1 arrest, activation of caspase-3, induction of Bax, inhibition of Bcl-2 and cleavage of poly (ADP-ribose) polymerase (PARP). In addition, Tob1 inhibited proliferation, migration and invasion, which were reversed in MKN1 and AGS cells transfected with Tob1 siRNA. Overexpression of Tob1 in MKN28 and AGS cells induced the expression of Smad4, leading to the increased expression and the promoter activity of p15, which was diminished by silencing of Tob1 using specific siRNA. Tob1 decreased the phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β) in MKN28 and AGS cells, resulting in the reduced protein expression and the transcriptional activity of β‑catenin, which in turn decreased the expression of cyclin D1, cyclin-dependent kinase-4 (CDK4), urokinase plasminogen activator receptor (uPAR) and peroxisome proliferator and activator receptor-δ (PPARδ). Conversely, silencing of Tob1 induced the phosphorylation of Akt and GSK-3β, and increased the expression of β‑catenin and its target genes. Collectively, our study demonstrates that the overexpression of Tob1 inhibits gastric cancer progression by activating Smad4- and inhibiting β‑catenin-mediated signaling pathways.

Fibrin-Enhanced Canonical Wnt Signaling Directs Plasminogen Expression in Cementoblasts

PubMed Central

Rahman, Saeed Ur; Ryoo, Hyun-Mo

2017-01-01

Cementum is a mineralized layer on the tooth’s root surface and facilitates the biomechanical anchoring of fibrous connective tissues as a part of tooth-supportive complexes. Previously, we observed that OCCM30 cementoblasts cultured on fibrin matrices underwent apoptosis due to fibrin degradation through the expression of proteases. Here, we demonstrated that OCCM30 on fibrin matrices (OCCM30-fibrin) enhanced canonical Wnt signaling, which directed to plasminogen expression. The OCCM30-fibrin showed higher levels of Wnt3a expression, nuclear translocation of β-catenin, and T-cell factor (TCF) optimal motif (TOP) reporter activity than the cells on tissue culture dishes (OCCM30-TCD), indicating that the OCCM30-fibrin enhanced canonical Wnt/β-catenin signaling. Also, OCCM30-fibrin expressed biomineralization-associated markers at higher levels than OCCM30-TCD, of which levels were further increased with LiCl, a Wnt signaling activator. The OCCM30 cementoblasts simultaneously showed that high levels of plasminogen, a critical component of fibrinolysis, were expressed in the OCCM30-fibrin. Activation of canonical Wnt signaling with LiCl treatment or with forced lymphoid enhancer factor 1 (LEF1)-expression increased the expression of plasminogen. On the contrary, the inhibition of canonical Wnt signaling with siRNAs against Wnt3a or β-catenin abrogated fibrin-enhanced plasminogen expression. Furthermore, there are three conserved putative response elements for the LEF1/β-catenin complex in the plasminogen proximal promoter regions (−900 to +54). Site-directed mutations and chromatin immunoprecipitation indicated that canonical Wnt signaling directed plasminogen expression. Taken together, this study suggests that fibrin-based materials can modulate functional periodontal formations in controlling cementoblast differentiation and fibrin degradation. PMID:29120400

Knockdown of Pentraxin 3 suppresses tumorigenicity and metastasis of human cervical cancer cells.

PubMed

Ying, Tsung-Ho; Lee, Chien-Hsing; Chiou, Hui-Ling; Yang, Shun-Fa; Lin, Chu-Liang; Hung, Chia-Hung; Tsai, Jen-Pi; Hsieh, Yi-Hsien

2016-07-05

Pentraxin 3 (PTX3) as an inflammatory molecule has been shown to be involved in immune response, inflammation, and cancer. However, the effects of PTX3 on the biological features of cervical cancer cells in vitro and in vivo have not been delineated. Immunohistochemical staining showed that increased PTX3 expression was significantly associated with tumor grade (P < 0.011) and differentiation (P < 0.019). Knocking down PTX3 with lentivirus-mediated small hairpin RNA (shRNA) in cervical cancer cell lines resulted in inhibited cell viability, diminished colony-forming ability, and induced cell cycle arrest at the G2/M phase of the cell cycle, along with downregulated expression of cyclin B1, cdc2, and cdc25c, and upregulated expression of p-cdc2, p-cdc25c, p21, and p27. Furthermore, knockdown of PTX3 significantly decreased the potential of migration and invasion of cervical cancer cells by inhibiting matrix metalloproteidase-2 (MMP-2), MMP-9, and urokinase plasminogen activator (uPA). Moreover, in vivo functional studies showed PTX3-knockdown in mice suppressed tumorigenicity and lung metastatic potential. Conversely, overexpression of PTX3 enhanced proliferation and invasion both in vitro and in vivo. Our results demonstrated that PTX3 contributes to tumorigenesis and metastasis of human cervical cancer cells. Further studies are warranted to demonstrate PTX3 as a novel therapeutic biomarker for human cervical cancer.

Safety, dose, and timing of reteplase in treating occluded central venous catheters in children with cancer.

PubMed

Terrill, Kelly R; Lemons, Richard S; Goldsby, Robert E

2003-11-01

Recombinant tissue plasminogen activator, alteplase, began to be commonly used to restore the patency of occluded central venous catheters (CVCs) as urokinase production was halted in the late 1990s. However, alteplase often requires an extended dwell time to restore patency to occluded CVCs. In adults, reteplase, a newer thrombolytic agent, has been reported to restore patency to CVCs in 30 minutes. The authors prospectively evaluated the safety and efficacy of reteplase in restoring patency to occluded CVCs in children with cancer. This was a dose escalation trial. The dose of reteplase was initiated at 0.1 units and increased by increments of 0.1 units to a maximum dose of 0.4 units. Each dose was tested on at least three participants. Time to patency after reteplase administration was recorded by nurses caring for the patients. Attempts to access the line occurred every 15 minutes for 1 hour. CVCs that remained occluded after 1 hour were treated with alteplase. Reteplase was administered to 15 clotted CVCs. Twelve of the 15 were cleared with an average dwell time of 38 minutes. The time to patency did not appear to correlate with the dose. No adverse events were reported. Reteplase can restore patency to occluded CVCs in a pediatric population. Reteplase appears to have comparable efficacy with alteplase, but reteplase may require shorter dwell times. A prospective, randomized, clinical trial is warranted to determine whether reteplase is as effective as alteplase in restoring patency to occluded CVCs.

Penicillin binding protein 3 of Staphylococcus aureus NCTC 8325-4 binds and activates human plasminogen.

PubMed

Kylväjä, Riikka; Ojalehto, Tuomas; Kainulainen, Veera; Virkola, Ritva; Westerlund-Wikström, Benita

2016-08-04

Staphylococcus aureus is a versatile pathogen expressing a number of virulence-associated adhesive molecules. In a previous study, we generated in a secretion-competent Escherichia coli strain a library of random FLAG-tag positive (FTP) polypeptides of S. aureus. To identify adhesive proteins and gain additional knowledge on putative virulence factors of S. aureus, we here screened the FTP library against human serum proteins. Staphylococcus aureus NCTC 8325-4, origin of the FTP library, adhered to immobilized plasminogen in vitro. In an enzyme-linked immunoassay a C-terminal part of penicillin binding protein 3 (PBP3), included in the FTP library, bound to immobilized plasminogen. We expressed and purified full-length PBP3 and its C-terminal fragments as recombinant proteins. In a time-resolved fluorometry-based assay the PBP3 polypeptides bound to immobilized plasminogen. The polypeptides enhanced formation of plasmin from plasminogen as analyzed by cleavage of a chromogenic plasmin substrate. The present findings, although preliminary, demonstrate reliably that S. aureus NCTC 8325-4 adheres to immobilized plasminogen in vitro and that the adhesion may be mediated by a C-terminal fragment of the PBP3 protein. The full length PBP3 and the penicillin binding C-terminal domain of PBP3 expressed as recombinant proteins bound plasminogen and activated plasminogen to plasmin. These phenomena were inhibited by the lysine analogue ε-aminocaproic acid suggesting that the binding is mediated by lysine residues. A detailed molecular description of surface molecules enhancing the virulence of S. aureus will aid in understanding of its pathogenicity and help in design of antibacterial drugs in the future.

u-PAR expression in cancer associated fibroblast: new acquisitions in multiple myeloma progression.

PubMed

Ciavarella, S; Laurenzana, A; De Summa, S; Pilato, B; Chillà, A; Lacalamita, R; Minoia, C; Margheri, F; Iacobazzi, A; Rana, A; Merchionne, F; Fibbi, G; Del Rosso, M; Guarini, A; Tommasi, S; Serratì, S

2017-03-24

Multiple Myeloma (MM) is a B-cell malignancy in which clonal plasma cells progressively expand within the bone marrow (BM) as effect of complex interactions with extracellular matrix and a number of microenvironmental cells. Among these, cancer-associated fibroblasts (CAF) mediate crucial reciprocal signals with MM cells and are associated to aggressive disease and poor prognosis. A large body of evidence emphasizes the role of the urokinase plasminogen activator (u-PA) and its receptor u-PAR in potentiating the invasion capacity of tumor plasma cells, but little is known about their role in the biology of MM CAF. In this study, we investigated the u-PA/u-PAR axis in MM-associated fibroblasts and explore additional mechanisms of tumor/stroma interplay in MM progression. CAF were purified from total BM stromal fraction of 64 patients including monoclonal gammopathy of undetermined significance, asymptomatic and symptomatic MM, as well as MM in post-treatment remission. Flow cytometry, Real Time PCR and immunofluorescence were performed to investigate the u-PA/u-PAR system in relation to the level of activation of CAF at different stages of the disease. Moreover, proliferation and invasion assays coupled with silencing experiments were used to prove, at functional level, the function of u-PAR in CAF. We found higher activation level, along with increased expression of pro-invasive molecules, including u-PA, u-PAR and metalloproteinases, in CAF from patients with symptomatic MM compared to the others stages of the disease. Consistently, CAF from active MM as well as U266 cell line under the influence of medium conditioned by active MM CAF, display higher proliferative rate and invasion potential, which were significantly restrained by u-PAR gene expression inhibition. Our data suggest that the stimulation of u-PA/u-PAR system contributes to the activated phenotype and function of CAF during MM progression, providing a biological rationale for future targeted therapies against MM.

Prognostic value of tissue-type plasminogen activator (tPA) and its complex with the type-1 inhibitor (PAI-1) in breast cancer

PubMed Central

Witte, J H de; Sweep, C G J; Klijn, J G M; Grebenschikov, N; Peters, H A; Look, M P; Tienoven, ThH van; Heuvel, J J T M; Vries, J Bolt-De; Benraad, ThJ; Foekens, J A

1999-01-01

The prognostic value of tissue-type plasminogen activator (tPA) measured in samples derived from 865 patients with primary breast cancer using a recently developed enzyme-linked immunosorbent assay (ELISA) was evaluated. Since the assay could easily be adapted to the assessment of the complex of tPA with its type-1 inhibitor (PAI-1), it was investigated whether the tPA:PAI-1 complex also provides prognostic information. To this end, cytosolic extracts and corresponding detergent extracts of 100 000 g pellets obtained after ultracentrifugation when preparing the cytosolic fractions for routine steroid hormone receptor determination were assayed. Statistically significant correlations were found between the cytosolic levels and those determined in the pellet extracts (Spearman correlation coefficient rs = 0.75, P < 0.001 for tPA and r = 0.50, P < 0.001 for tPA:PAI-1 complex). In both Cox univariate and multivariate analysis elevated levels of (total) tPA determined in the pellet extracts, but not in cytosols, were associated with prolonged relapse-free (RFS) and overall survival (OS). In contrast, high levels of the tPA:PAI-1 complex measured in cytosols, but not in the pellet extracts, were associated with a poor RFS and OS. The prognostic information provided by the cytosolic tPA:PAI-1 complex was comparable to that provided by cytosolic (total) PAI-1. Furthermore, the estimated levels of free, uncomplexed tPA and PAI-1, in cytosols and in pellet extracts, were related to patient prognosis in a similar way as the (total) levels of tPA and PAI-1 respectively. Determination of specific forms of components of the plasminogen activation system, i.e. tPA:PAI-1 complex and free, uncomplexed tPA and/or PAI-1, may be considered a useful adjunct to the analyses of the separate components (tPA and/or PAI-1) and provide valuable additional prognostic information with respect to survival of breast cancer patients. © 1999 Cancer Research Campaign PMID:10390010

Influences of Ivabradine treatment on serum levels of cardiac biomarkers sST2, GDF-15, suPAR and H-FABP in patients with chronic heart failure.

PubMed

Jirak, Peter; Fejzic, Dzeneta; Paar, Vera; Wernly, Bernhard; Pistulli, Rudin; Rohm, Ilonka; Jung, Christian; Hoppe, Uta C; Schulze, P Christian; Lichtenauer, Michael; Yilmaz, Atilla; Kretzschmar, Daniel

2017-12-14

Chronic heart failure (CHF) represents a major cause of hospitalization and death. Recent evidence shows that novel biomarkers such as soluble suppression of tumorigenicity (sST2), growth-differentiation factor-15 (GDF-15), soluble urokinase plasminogen activator receptor (suPAR) and heart-type fatty acid binding protein (H-FABP) are correlated with inflammatory and ischemic responses in CHF patients. In this study we examined the effects of Ivabradine that inhibited the hyperpolarization-activated cyclic nucleotide-gated channel (HCN channel, also called funny current I f ), thereby leading to selective heart rate reduction and improved myocardial oxygen supply on the cardiac biomarkers sST2, GDF-15, suPAR and H-FABP in 50 CHF patients at the University Hospital of Jena. Patients were divided into three groups based on the etiology of CHF: dilated cardiomyopathy (DCM, n=20), ischemic cardiomyopathy (ICM, n=20) and hypertensive cardiomyopathy (HCM, n=10). The patients were administered Ivabradine (5 mg, bid for 3 months, and 7.5 mg bid for further 3 months). Analyses of cardiovascular biomarkers were performed at baseline as well as at 3- and 6-month follow-ups. At 6-month follow-up, GDF-15 levels were significantly reduced compared to baseline levels (P=0.0215), indicating a reduction in the progress of cardiac remodeling. H-FABP concentration was significantly lower in DCM patients compared to ICM (1.89 vs 3.24 μg/mL) and HCM patients (1.89 vs 3.80 μg/mL), and decreased over the 6-month follow-up (P=0.0151). suPAR median levels remained elevated, implying major ongoing inflammatory processes. As shown by significant decreases in GDF-15 and H-FABP levels, a reduction in ventricular remodeling and sub-clinical ischemia could be assumed. However, markers of hemodynamic stress (sST2) and inflammation (suPAR) showed no change or progression after 6 months of Ivabradine treatment in CHF patients. Further studies are necessary to validate the clinical applicability of these novel cardiovascular biomarkers.

The duplication mutation of Quebec platelet disorder dysregulates PLAU, but not C10orf55, selectively increasing production of normal PLAU transcripts by megakaryocytes but not granulocytes.

PubMed

Hayward, Catherine P M; Liang, Minggao; Tasneem, Subia; Soomro, Asim; Waye, John S; Paterson, Andrew D; Rivard, Georges E; Wilson, Michael D

2017-01-01

Quebec Platelet disorder (QPD) is a unique bleeding disorder that markedly increases urokinase plasminogen activator (uPA) in megakaryocytes and platelets but not in plasma or urine. The cause is tandem duplication of a 78 kb region of chromosome 10 containing PLAU (the uPA gene) and C10orf55, a gene of unknown function. QPD increases uPA in platelets and megakaryocytes >100 fold, far more than expected for a gene duplication. To investigate the tissue-specific effect that PLAU duplication has on gene expression and transcript structure in QPD, we tested if QPD leads to: 1) overexpression of normal or unique PLAU transcripts; 2) increased uPA in leukocytes; 3) altered levels of C10orf55 mRNA and/or protein in megakaryocytes and leukocytes; and 4) global changes in megakaryocyte gene expression. Primary cells and cultured megakaryocytes from donors were prepared for quantitative reverse polymerase chain reaction analyses, RNA-seq and protein expression analyses. Rapidly isolated blood leukocytes from QPD subjects showed only a 3.9 fold increase in PLAU transcript levels, in keeping with the normal to minimally increased uPA in affinity purified, QPD leukocytes. All subjects had more uPA in granulocytes than monocytes and minimal uPA in lymphocytes. QPD leukocytes expressed PLAU alleles in proportions consistent with an extra copy of PLAU on the disease chromosome, unlike QPD megakaryocytes. QPD PLAU transcripts were consistent with reference gene models, with a much higher proportion of reads originating from the disease chromosome in megakaryocytes than granulocytes. QPD and control megakaryocytes contained minimal reads for C10orf55, and C10orf55 protein was not increased in QPD megakaryocytes or platelets. Finally, our QPD megakaryocyte transcriptome analysis revealed a global down regulation of the interferon type 1 pathway. We suggest that the low endogenous levels of uPA in blood are actively regulated, and that the regulatory mechanisms are disrupted in QPD in a megakaryocyte-specific manner.

The duplication mutation of Quebec platelet disorder dysregulates PLAU, but not C10orf55, selectively increasing production of normal PLAU transcripts by megakaryocytes but not granulocytes

PubMed Central

Soomro, Asim; Waye, John S.; Paterson, Andrew D.; Rivard, Georges E.; Wilson, Michael D.

2017-01-01

Quebec Platelet disorder (QPD) is a unique bleeding disorder that markedly increases urokinase plasminogen activator (uPA) in megakaryocytes and platelets but not in plasma or urine. The cause is tandem duplication of a 78 kb region of chromosome 10 containing PLAU (the uPA gene) and C10orf55, a gene of unknown function. QPD increases uPA in platelets and megakaryocytes >100 fold, far more than expected for a gene duplication. To investigate the tissue-specific effect that PLAU duplication has on gene expression and transcript structure in QPD, we tested if QPD leads to: 1) overexpression of normal or unique PLAU transcripts; 2) increased uPA in leukocytes; 3) altered levels of C10orf55 mRNA and/or protein in megakaryocytes and leukocytes; and 4) global changes in megakaryocyte gene expression. Primary cells and cultured megakaryocytes from donors were prepared for quantitative reverse polymerase chain reaction analyses, RNA-seq and protein expression analyses. Rapidly isolated blood leukocytes from QPD subjects showed only a 3.9 fold increase in PLAU transcript levels, in keeping with the normal to minimally increased uPA in affinity purified, QPD leukocytes. All subjects had more uPA in granulocytes than monocytes and minimal uPA in lymphocytes. QPD leukocytes expressed PLAU alleles in proportions consistent with an extra copy of PLAU on the disease chromosome, unlike QPD megakaryocytes. QPD PLAU transcripts were consistent with reference gene models, with a much higher proportion of reads originating from the disease chromosome in megakaryocytes than granulocytes. QPD and control megakaryocytes contained minimal reads for C10orf55, and C10orf55 protein was not increased in QPD megakaryocytes or platelets. Finally, our QPD megakaryocyte transcriptome analysis revealed a global down regulation of the interferon type 1 pathway. We suggest that the low endogenous levels of uPA in blood are actively regulated, and that the regulatory mechanisms are disrupted in QPD in a megakaryocyte-specific manner. PMID:28301587

Mannose Receptor 2 Attenuates Renal Fibrosis

PubMed Central

López-Guisa, Jesús M.; Cai, Xiaohe; Collins, Sarah J.; Yamaguchi, Ikuyo; Okamura, Daryl M.; Bugge, Thomas H.; Isacke, Clare M.; Emson, Claire L.; Turner, Scott M.; Shankland, Stuart J.

2012-01-01

Mannose receptor 2 (Mrc2) expresses an extracellular fibronectin type II domain that binds to and internalizes collagen, suggesting that it may play a role in modulating renal fibrosis. Here, we found that Mrc2 levels were very low in normal kidneys but subsets of interstitial myofibroblasts and macrophages upregulated Mrc2 after unilateral ureteral obstruction (UUO). Renal fibrosis and renal parenchymal damage were significantly worse in Mrc2-deficient mice. Similarly, Mrc2-deficient Col4α3−/− mice with hereditary nephritis had significantly higher levels of total kidney collagen, serum BUN, and urinary protein than Mrc2-sufficient Col4α3−/− mice. The more severe phenotype seemed to be the result of reduced collagen turnover, because procollagen III (α1) mRNA levels and fractional collagen synthesis in the wild-type and Mrc2-deficient kidneys were similar after UUO. Although Mrc2 associates with the urokinase receptor, differences in renal urokinase activity did not account for the increased fibrosis in the Mrc2-deficient mice. Treating wild-type mice with a cathepsin inhibitor, which blocks proteases implicated in Mrc2-mediated collagen degradation, worsened UUO-induced renal fibrosis. Cathepsin mRNA profiles were similar in Mrc2-positive fibroblasts and macrophages, and Mrc2 genotype did not alter relative cathepsin mRNA levels. Taken together, these data establish an important fibrosis-attenuating role for Mrc2-expressing renal interstitial cells and suggest the involvement of a lysosomal collagen turnover pathway. PMID:22095946

Compaction of fibrin clots reveals the antifibrinolytic effect of factor XIII.

PubMed

Rijken, D C; Abdul, S; Malfliet, J J M C; Leebeek, F W G; Uitte de Willige, S

2016-07-01

Essentials Factor XIIIa inhibits fibrinolysis by forming fibrin-fibrin and fibrin-inhibitor cross-links. Conflicting studies about magnitude and mechanisms of inhibition have been reported. Factor XIIIa most strongly inhibits lysis of mechanically compacted or retracted plasma clots. Cross-links of α2-antiplasmin to fibrin prevent the inhibitor from being expelled from the clot. Background Although insights into the underlying mechanisms of the effect of factor XIII on fibrinolysis have improved considerably in the last few decades, in particular with the discovery that activated FXIII (FXIIIa) cross-links α2 -antiplasmin to fibrin, the topic remains a matter of debate. Objective To elucidate the mechanisms of the antifibrinolytic effect of FXIII. Methods and Results Platelet-poor plasma clot lysis, induced by the addition of tissue-type plasminogen activator, was measured in the presence or absence of a specific FXIIIa inhibitor. Both in a turbidity assay and in a fluorescence assay, the FXIIIa inhibitor had only a small inhibitory effect: 1.6-fold less tissue-type plasminogen activator was required for 50% clot lysis in the presence of the FXIIIa inhibitor. However, when the plasma clot was compacted by centrifugation, the FXIIIa inhibitor had a strong inhibitory effect, with 7.7-fold less tissue-type plasminogen activator being required for 50% clot lysis in the presence of the FXIIIa inhibitor. In both experiments, the effects of the FXIIIa inhibitor were entirely dependent on the cross-linking of α2 -antiplasmin to fibrin. The FXIIIa inhibitor reduced the amount of α2 -antiplasmin present in the compacted clots from approximately 30% to < 4%. The results were confirmed with experiments in which compaction was achieved by platelet-mediated clot retraction. Conclusions Compaction or retraction of fibrin clots reveals the strong antifibrinolytic effect of FXIII. This is explained by the cross-linking of α2 -antiplasmin to fibrin by FXIIIa, which prevents the plasmin inhibitor from being fully expelled from the clot during compaction/retraction. © 2016 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.

[The effect of urokinase on hepatic fibrogenesis in rats].

PubMed

Wu, Xi-run; Wang, Qi; Wang, Ling; Shi, Shui-sheng; Guo, Wen-dong

2009-12-01

To investigate the effect of urokinase on hepatic fibrogenesis in rats. Hepatic fibrosis was induced in rats by complex pathogenic factors including subcutaneous injections of carbon tetrachloride, alcohol and cholesterol feeding. Animals were randomly divided into 3 groups: normal control group, hepatic fibrosis group (complex pathogenic factors for 6 weeks), UK prevention group (complex pathogenic factors+UK for 6 weeks). The animals were sacrificed at the end of week 6. The expression of alpha-SMA, uPA, PAI-1, TGFb1, TIMP-1, collagen type I and type III proteins in hepatic fibrosis tissue was detected by immunohistochemistry, the expression of PAI-1 and TGFb1 mRNA in the hepatic fibrosis tissue was quantified by real time RT-PCR. The serum levels of hyaluronicacid (HA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin (TBil) and the content of liver hydroxyproline (Hyp) were detected using ELISA kits. The serum ALT, AST, TBil, HA and the content of liver Hyp were (46.66+/-6.30) U/L, (126.26+/-31.65) U/L, (31.11+/-4.20) micromol/L, (109.70+/-18.81) microg/L and (0.98+/-0.09) mg/(g liver), respectively, in UK prevention group, which were significantly lower than those [(101.57+/-11.97) U/L, (205.89+/-56.26) U/L, (67.75+/-2.75) micromol/L, (184.43+/-32.36) microg/L and (1.65+/-0.16) mg/(g liver), respectively] in hepatic fibrosis group (q = 3.3801-20.0061, P < 0.01). The levels of a-SMA, collagen type I, type III, TIMP-1, PAI-1, TGFb1 proteins were (299.27+/-37.36), (210.05+/-27.17), (192.94+/-24.48), (213.70+/-32.21), (204.25+/-17.92), (205.97+/-23.81), respectively, in UK prevention group, which were significantly lower than those [(418.83+/-30.21), (323.77+/-21.53), (302.37+/-31.43), (376.63+/-25.19), (313.53+/-26.67) and (327.42+/-36.75), respectively] in hepatic fibrosis group. The level of uPA protein was increased, and the expression of PAI-1, TGFb1 mRNA in hepatic fibrosis tissue was decreased in UK prevention group. In the early stage of hepatic fibrogenesis, urokinase can attenuate the progression of rat hepatic fibrosis via upregulation of uPA, downregulation of TGFb1, and inhibition of HSC activation.

Lingual Haematoma due to Tenecteplase in a Patient with Acute Myocardial Infarction

PubMed Central

Bal, Muhlis; Salturk, Ziya; Ateş, Ahmet Hakan; Yağcı, Serkan; Coşkun Bal, Gökçen

2013-01-01

The use of intravenous thrombolytic agents has revolutionised the treatment of acute myocardial infarction. However, the improvement in mortality rate achieved with these drugs is tempered by the risk of serious bleeding complications, including intracranial haemorrhage. Tenecteplase is a genetically engineered mutant tissue plasminogen activator. Haemorrhagic complications of tissue plasminogen activator (tPA) are well known. Compared to other tPAs, tenecteplase use leads to lower rates of bleeding complications. Here, we report a case of unusual site of spontaneous bleeding, intralingual haematoma during tenecteplase therapy following acute myocardial infarction, which caused significant upper airway obstruction and required tracheotomy to maintain the patient's airway. Clinical dilemmas related to securing the airway or reversing the effects of tissue plasminogen activator are discussed. PMID:23862086

Subunits of the Pyruvate Dehydrogenase Cluster of Mycoplasma pneumoniae Are Surface-Displayed Proteins that Bind and Activate Human Plasminogen

PubMed Central

Gründel, Anne; Friedrich, Kathleen; Pfeiffer, Melanie; Jacobs, Enno; Dumke, Roger

2015-01-01

The dual role of glycolytic enzymes in cytosol-located metabolic processes and in cell surface-mediated functions with an influence on virulence is described for various micro-organisms. Cell wall-less bacteria of the class Mollicutes including the common human pathogen Mycoplasma pneumoniae possess a reduced genome limiting the repertoire of virulence factors and metabolic pathways. After the initial contact of bacteria with cells of the respiratory epithelium via a specialized complex of adhesins and release of cell-damaging factors, surface-displayed glycolytic enzymes may facilitate the further interaction between host and microbe. In this study, we described detection of the four subunits of pyruvate dehydrogenase complex (PDHA-D) among the cytosolic and membrane-associated proteins of M. pneumoniae. Subunits of PDH were cloned, expressed and purified to produce specific polyclonal guinea pig antisera. Using colony blotting, fractionation of total proteins and immunofluorescence experiments, the surface localization of PDHA-C was demonstrated. All recombinant PDH subunits are able to bind to HeLa cells and human plasminogen. These interactions can be specifically blocked by the corresponding polyclonal antisera. In addition, an influence of ionic interactions on PDHC-binding to plasminogen as well as of lysine residues on the association of PDHA-D with plasminogen was confirmed. The PDHB subunit was shown to activate plasminogen and the PDHB-plasminogen complex induces degradation of human fibrinogen. Hence, our data indicate that the surface-associated PDH subunits might play a role in the pathogenesis of M. pneumoniae infections by interaction with human plasminogen. PMID:25978044

No difference in portal and hepatic venous bacterial DNA in patients with cirrhosis undergoing transjugular intrahepatic portosystemic shunt insertion.

PubMed

Mortensen, Christian; Karlsen, Stine; Grønbæk, Henning; Nielsen, Dennis T; Frevert, Susanne; Clemmesen, Jens O; Møller, Søren; Jensen, Jørgen S; Bendtsen, Flemming

2013-10-01

Bacterial translocation (BT) with immune activation may lead to hemodynamical alterations and poor outcomes in patients with cirrhosis. We investigated bacterial DNA (bDNA), a marker of BT, and its relation to portal pressure and markers of inflammation in the portal and hepatic veins in patients with cirrhosis undergoing TIPS insertion. We analysed plasma for bDNA and markers of inflammation in 28 patients [median portal pressure gradient 15 (11-19) mmHg] during TIPS treatment for refractory ascites (n = 19) or acute variceal bleeding (n = 9). Advanced cirrhosis was present in the majority [Child-Pugh class (A/B/C): 1/14/13], and most often caused by alcohol (n = 21). bDNA was detectable in one or both samples in 16 of 28 patients (57%). bDNA was present in 39% of the samples from the portal vein vs 43% of the samples in the hepatic vein (P = 0.126). Antibiotics had no effect on bDNA or markers of inflammation. Markers of inflammation did not differ between the hepatic and portal veins with the exceptions of soluble urokinase plasminogen activating receptor (suPAR) and vascular endothelial growth factor (VEGF), both higher in the hepatic vein (P = 0.031 and 0.003 respectively). No transhepatic gradient of bDNA was evident, suggesting that no major hepatic elimination of bDNA occurs in advanced liver disease. bDNA, in contrast to previous reports was largely unrelated to a panel of markers of inflammation and without relation to portal pressure. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Uninvolved Skin from Psoriatic Patients Develops Signs of Involved Psoriatic Skin after Being Grafted onto Nude Mice

NASA Astrophysics Data System (ADS)

Fraki, Jorma E.; Briggaman, Robert A.; Lazarus, Gerald S.

1982-02-01

Clinically involved psoriatic epidermis maintains its histological appearance, increased labeling index, and increased level of plasminogen activator after being grafted onto athymic nude mice. Uninvolved psoriatic epidermis develops increases in plasminogen activator activity after being grafted onto athymic nude mice; this is accompanied by an increased labeling index. Thus, psoriatic skin can develop markers of psoriasis independent of the host.

Proposal for the nomenclature of human plasminogen (PLG) polymorphism.

PubMed

Skoda, U; Bertrams, J; Dykes, D; Eiberg, H; Hobart, M; Hummel, K; Kühnl, P; Mauff, G; Nakamura, S; Nishimukai, H

1986-01-01

Since its discovery, human plasminogen (PLG) polymorphism has received widespread acceptance in population genetics and forensic haematology. Due to the large number of variant alleles described, a PLG reference typing and Plasminogen Symposium was held, at which a nomenclature proposal was inaugurated. The technology of comparing PLG variants was based on isoelectric focusing and subsequent detection by caseinolytic overlay and 'Western' blotting. Typing results permitted comparison of so far described variant designations and resulted in a new nomenclature proposal for PLG polymorphism. It is recommended that the two most common alleles found in all investigated races be called: PLG*A (previously also PLG*1) and PLG*B (previously also PLG*2), the known variants with acidic pI: PLG*A1 to *A3, intermediate variants: PLG*M1 to *M5, PLG*M5 being functionally inactive, and basic variants: PLG*B1 to *B3. For future classification of newly discovered variants, samples should be compared at any of the laboratories participating in the reference typing.

MicroRNA expression profile in endometriosis: its relation to angiogenesis and fibrinolytic factors.

PubMed

Braza-Boïls, Aitana; Marí-Alexandre, Josep; Gilabert, Juan; Sánchez-Izquierdo, Dolors; España, Francisco; Estellés, Amparo; Gilabert-Estellés, Juan

2014-05-01

Could an aberrant microRNA (miRNA) expression profile be responsible for the changes in the angiogenic and fibrinolytic states observed in endometriotic lesions? This study revealed characteristic miRNA expression profiles associated with endometriosis in endometrial tissue and endometriotic lesions from the same patient and their correlation with the most important angiogenic and fibrinolytic factors. WHAT IS ALREADY KNOWN?: An important role for dysregulated miRNA expression in the pathogenesis of endometriosis is well documented. However, to the best of our knowledge, there are no reports of the relationship between angiogenic and fibrinolytic factors and miRNAs when endometrial tissue and different types of endometriotic lesions from the same patient are compared. Case-control study that involved 51 women with endometriosis and 32 women without the disease (controls). The miRNA expression profiles were determined using the GeneChip miRNA 2.0 Affymetrix array platform, and the results were analysed using Partek Genomic Suite software. To validate the obtained results, 12 miRNAs differentially expressed were quantified by using miRCURY LNA™ Universal RT microRNA PCR. Levels of vascular endothelial growth factor (VEGF-A), thrombospondin-1 (TSP-1), urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) proteins were quantified by ELISA. Patient endometrial tissue showed significantly lower levels of miR-202-3p, miR-424-5p, miR-449b-3p and miR-556-3p, and higher levels of VEGF-A and uPA than healthy (control) endometrium. However, tissue affected by ovarian endometrioma showed significantly lower expression of miR-449b-3p than endometrium from both controls and patients, and higher levels of PAI-1 and the angiogenic inhibitor TSP-1. A significant inverse correlation between miR-424-5p and VEGF-A protein levels was observed in patient endometrium, and an inverse correlation between miR-449b-3p and TSP-1 protein levels was observed in ovarian endometrioma. Peritoneal implants had significantly higher levels of VEGF-A than ovarian endometrioma samples. Functional studies are needed to confirm the specific targets of the miRNAs differently expressed. Differences in miRNA levels could modulate the expression of VEGF-A and TSP-1, which may play an important role in the pathogenesis of endometriosis. The higher angiogenic and proteolytic activities observed in eutopic endometrium from patients might facilitate the implantation of endometrial cells at ectopic sites. This work was supported by research grants from ISCIII-FEDER (PI11/0091, Red RIC RD12/0042/0029), Consellería de Educación-Generalitat Valenciana (PROMETEO/2011/027), Beca de Investigación Fundación Dexeus para la Salud de la Mujer (2011/0469), and by Fundación Investigación Hospital La Fe (2011/211). A.B-B. has a Contrato Posdoctoral de Perfeccionamiento Sara Borrell-ISCIII (CD13/00005). J.M-A. has a predoctoral grant PFIS-ISCIII (FI12/00012). The authors have no conflicts of interest to declare.

Comparison between the clot-protecting activity of a mutant plasminogen activator inhibitor-1 with a very long half-life and 6-aminocaproic acid.

PubMed

Kindell, Daniel Glenn; Keck, Rick Wayne; Jankun, Jerzy

2015-06-01

Plasminogen activator inhibitor (PAI)-1 is a serpin glycoprotein that can stabilize blood clots by inhibiting fibrinolysis. However, wild-type PAI-1 has the disadvantage of a short half-life of ∼2 h. A very long half-life (VLHL) PAI-1 mutant was developed previously with an active-form half-life of >700 h, making it a possible candidate for use in hemorrhagic therapy. Current treatments for mitigating hemorrhage, other than inducers of blood clotting, are limited to lysine analog antifibrinolytics, including 6-aminocaproic acid and tranexamic acid. VLHL PAI-1 has been previously demonstrated to limit bleeding; however, the efficacy of this protein compared with lysine analog antifibrinolytics has not been investigated. The aim of the current study was to compare the clot stabilizing properties of the novel antifibrinolytic VLHL PAI-1 with those of 6-aminocaproic acid in reference plasma. Using thromboelastographic analysis, VLHL PAI-1 exhibited an IC 50 (half maximal inhibitory concentration) of 8.8×10 -8 mol/l, while 6-aminocaproic acid showed an IC 50 of 1.6×10 -4 mol/l. However, at doses of >9.0×10 -7 mol/l, VLHL PAI-1 exhibited a delay in the onset of clot formation, which may be attributed to thrombin inhibition by excess PAI-1. The inhibition of tissue plasminogen activator by VLHL PAI-1 demonstrated improved efficacy over 6-aminocaproic acid in mitigating hemorrhage. In addition, patients with a PAI-1 deficiency, which causes blood clots to lyse rapidly resulting in profuse bleeding, may benefit from the application of VLHL PAI-1 as an antihemorrhagic therapy.

Comparison between the clot-protecting activity of a mutant plasminogen activator inhibitor-1 with a very long half-life and 6-aminocaproic acid

PubMed Central

KINDELL, DANIEL GLENN; KECK, RICK WAYNE; JANKUN, JERZY

2015-01-01

Plasminogen activator inhibitor (PAI)-1 is a serpin glycoprotein that can stabilize blood clots by inhibiting fibrinolysis. However, wild-type PAI-1 has the disadvantage of a short half-life of ∼2 h. A very long half-life (VLHL) PAI-1 mutant was developed previously with an active-form half-life of >700 h, making it a possible candidate for use in hemorrhagic therapy. Current treatments for mitigating hemorrhage, other than inducers of blood clotting, are limited to lysine analog antifibrinolytics, including 6-aminocaproic acid and tranexamic acid. VLHL PAI-1 has been previously demonstrated to limit bleeding; however, the efficacy of this protein compared with lysine analog antifibrinolytics has not been investigated. The aim of the current study was to compare the clot stabilizing properties of the novel antifibrinolytic VLHL PAI-1 with those of 6-aminocaproic acid in reference plasma. Using thromboelastographic analysis, VLHL PAI-1 exhibited an IC50 (half maximal inhibitory concentration) of 8.8×10−8 mol/l, while 6-aminocaproic acid showed an IC50 of 1.6×10−4 mol/l. However, at doses of >9.0×10−7 mol/l, VLHL PAI-1 exhibited a delay in the onset of clot formation, which may be attributed to thrombin inhibition by excess PAI-1. The inhibition of tissue plasminogen activator by VLHL PAI-1 demonstrated improved efficacy over 6-aminocaproic acid in mitigating hemorrhage. In addition, patients with a PAI-1 deficiency, which causes blood clots to lyse rapidly resulting in profuse bleeding, may benefit from the application of VLHL PAI-1 as an antihemorrhagic therapy. PMID:26136983

Regulatory elements involved in constitutive and phorbol ester-inducible expression of the plasminogen activator inhibitor type 2 gene promoter.

PubMed Central

Cousin, E; Medcalf, R L; Bergonzelli, G E; Kruithof, E K

1991-01-01

Gene transcription rates and mRNA levels of plasminogen activator inhibitor type 2 (PAI-2) are markedly induced by the tumor promoting agent phorbol 12-myristate 13-acetate (PMA) in human HT1080 fibrosarcoma cells. To identify promoter elements required for basal-, and phorbol ester-inducible expression, deletion mutants of the PAI-1 promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene, were transiently expressed in HT1080 cells. Constitutive CAT activity was expressed from constructs containing more than 215 bp of promoter sequence, whereas deletion to position -91 bp abolished CAT gene expression. Treatment of transfected cells with PMA resulted in a three- to ten-fold increase in CAT expression from all constructs except from the construct shortened to position -91. DNAse1 protection analysis of the promoter region between -215 and the transcription initiation site revealed numerous protected regions, including two AP1-like binding sites (AP1a and AP1b) and one CRE-like element. Site-directed mutagenesis of the AP1a site or of the CRE-like site resulted in the loss of basal CAT activity and abolished the PMA effect, whereas mutagenesis of AP1b only partially inhibited basal and PMA-mediated expression. Our results suggest that the PAI-2 promoter contains at least two elements required for basal gene transcription and PMA-mediated induction. Images PMID:1650454

Differential Regulation of PAI-1 in Hantavirus Cardiopulmonary Syndrome and Hemorrhagic Fever With Renal Syndrome.

PubMed

Bellomo, Carla; Korva, Miša; Papa, Anna; Mäkelä, Satu; Mustonen, Jukka; Avšič-Županc, Tatjana; Vaheri, Antti; Martinez, Valeria P; Strandin, Tomas

2018-02-01

We analyzed the levels of circulating tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI)-1 in acute hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS). The levels of tPA commonly increased in both diseases, whereas PAI-1 correlated with disease severity in HCPS but not in HFRS.

Dose dependency of outcomes of intrapleural fibrinolytic therapy in new rabbit empyema models

PubMed Central

Florova, Galina; Azghani, Ali O.; Buchanan, Ann; Boren, Jake; Allen, Timothy; Rahman, Najib M.; Koenig, Kathleen; Chamiso, Mignote; Karandashova, Sophia; Henry, James; Idell, Steven

2016-01-01

The incidence of empyema (EMP) is increasing worldwide; EMP generally occurs with pleural loculation and impaired drainage is often treated with intrapleural fibrinolytic therapy (IPFT) or surgery. A number of IPFT options are used clinically with empiric dosing and variable outcomes in adults. To evaluate mechanisms governing intrapleural fibrinolysis and disease outcomes, models of Pasteurella multocida and Streptococcus pneumoniae were generated in rabbits and the animals were treated with either human tissue (tPA) plasminogen activator or prourokinase (scuPA). Rabbit EMP was characterized by the development of pleural adhesions detectable by chest ultrasonography and fibrinous coating of the pleura. Similar to human EMP, rabbits with EMP accumulated sizable, 20- to 40-ml fibrinopurulent pleural effusions associated with extensive intrapleural organization, significantly increased pleural thickness, suppression of fibrinolytic and plasminogen-activating activities, and accumulation of high levels of plasminogen activator inhibitor 1, plasminogen, and extracellular DNA. IPFT with tPA (0.145 mg/kg) or scuPA (0.5 mg/kg) was ineffective in rabbit EMP (n = 9 and 3 for P. multocida and S. pneumoniae, respectively); 2 mg/kg tPA or scuPA IPFT (n = 5) effectively cleared S. pneumoniae-induced EMP collections in 24 h with no bleeding observed. Although intrapleural fibrinolytic activity for up to 40 min after IPFT was similar for effective and ineffective doses of fibrinolysin, it was lower for tPA than for scuPA treatments. These results demonstrate similarities between rabbit and human EMP, the importance of pleural fluid PAI-1 activity, and levels of plasminogen in the regulation of intrapleural fibrinolysis and illustrate the dose dependency of IPFT outcomes in EMP. PMID:27343192

Dose dependency of outcomes of intrapleural fibrinolytic therapy in new rabbit empyema models.

PubMed

Komissarov, Andrey A; Florova, Galina; Azghani, Ali O; Buchanan, Ann; Boren, Jake; Allen, Timothy; Rahman, Najib M; Koenig, Kathleen; Chamiso, Mignote; Karandashova, Sophia; Henry, James; Idell, Steven

2016-08-01

The incidence of empyema (EMP) is increasing worldwide; EMP generally occurs with pleural loculation and impaired drainage is often treated with intrapleural fibrinolytic therapy (IPFT) or surgery. A number of IPFT options are used clinically with empiric dosing and variable outcomes in adults. To evaluate mechanisms governing intrapleural fibrinolysis and disease outcomes, models of Pasteurella multocida and Streptococcus pneumoniae were generated in rabbits and the animals were treated with either human tissue (tPA) plasminogen activator or prourokinase (scuPA). Rabbit EMP was characterized by the development of pleural adhesions detectable by chest ultrasonography and fibrinous coating of the pleura. Similar to human EMP, rabbits with EMP accumulated sizable, 20- to 40-ml fibrinopurulent pleural effusions associated with extensive intrapleural organization, significantly increased pleural thickness, suppression of fibrinolytic and plasminogen-activating activities, and accumulation of high levels of plasminogen activator inhibitor 1, plasminogen, and extracellular DNA. IPFT with tPA (0.145 mg/kg) or scuPA (0.5 mg/kg) was ineffective in rabbit EMP (n = 9 and 3 for P. multocida and S. pneumoniae, respectively); 2 mg/kg tPA or scuPA IPFT (n = 5) effectively cleared S. pneumoniae-induced EMP collections in 24 h with no bleeding observed. Although intrapleural fibrinolytic activity for up to 40 min after IPFT was similar for effective and ineffective doses of fibrinolysin, it was lower for tPA than for scuPA treatments. These results demonstrate similarities between rabbit and human EMP, the importance of pleural fluid PAI-1 activity, and levels of plasminogen in the regulation of intrapleural fibrinolysis and illustrate the dose dependency of IPFT outcomes in EMP. Copyright © 2016 the American Physiological Society.

Intrapleural Adenoviral Delivery of Human Plasminogen Activator Inhibitor–1 Exacerbates Tetracycline-Induced Pleural Injury in Rabbits

PubMed Central

Karandashova, Sophia; Florova, Galina; Azghani, Ali O.; Komissarov, Andrey A.; Koenig, Kathy; Tucker, Torry A.; Allen, Timothy C.; Stewart, Kris; Tvinnereim, Amy

2013-01-01

Elevated concentrations of plasminogen activator inhibitor–1 (PAI-1) are associated with pleural injury, but its effects on pleural organization remain unclear. A method of adenovirus-mediated delivery of genes of interest (expressed under a cytomegalovirus promoter) to rabbit pleura was developed and used with lacZ and human (h) PAI-1. Histology, β-galactosidase staining, Western blotting, enzymatic and immunohistochemical analyses of pleural fluids (PFs), lavages, and pleural mesothelial cells were used to evaluate the efficiency and effects of transduction. Transduction was selective and limited to the pleural mesothelial monolayer. The intrapleural expression of both genes was transient, with their peak expression at 4 to 5 days. On Day 5, hPAI-1 (40–80 and 200–400 nM of active and total hPAI-1 in lavages, respectively) caused no overt pleural injury, effusions, or fibrosis. The adenovirus-mediated delivery of hPAI-1 with subsequent tetracycline-induced pleural injury resulted in a significant exacerbation of the pleural fibrosis observed on Day 5 (P = 0.029 and P = 0.021 versus vehicle and adenoviral control samples, respectively). Intrapleural fibrinolytic therapy (IPFT) with plasminogen activators was effective in both animals overexpressing hPAI-1 and control animals with tetracycline injury alone. An increase in intrapleural active PAI-1 (from 10–15 nM in control animals to 20–40 nM in hPAI-1–overexpressing animals) resulted in the increased formation of PAI-1/plasminogen activator complexes in vivo. The decrease in intrapleural plasminogen-activating activity observed at 10 to 40 minutes after IPFT correlates linearly with the initial concentration of active PAI-1. Therefore, active PAI-1 in PFs affects the outcome of IPFT, and may be both a biomarker of pleural injury and a molecular target for its treatment. PMID:23002099

Micronutrient Synergy in the Fight against Hepatocellular Carcinoma.

PubMed

Roomi, M Waheed; Roomi, Nusrath W; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

2012-03-23

The incidence of hepatocellular carcinoma (HCC), once thought to be a rare tumor in North America, has rapidly increased in recent years in the United States. Current treatment modalities to halt the progression of this disease are only marginally effective. The mainstay treatment is liver transplantation, which is often confronted with donor shortage. Invasion, metastasis and recurrence contribute to the high mortality rate of this disease. Matrix metalloproteinases (MMPs) that degrade the extracellular matrix (ECM) have been associated with the progression, invasion and metastasis of the disease. We have developed strategies to strengthen the ECM collagen and inhibit MMPs through micronutrients such as lysine, proline and ascorbic acid. Addition of epigallocatechin gallate or green tea extract to these micronutrients synergistically enhanced anti-carcinogenic activity in HepG2 cells. Addition of certain other micronutrients, such as N-acetylcysteine, selenium, copper and zinc (NM) synergistically enhanced the anticancer activity of the mixture in a model of hepatocellular carcinoma using HepG2 cells. In vitro studies using HepG2 demonstrated that NM was very effective in inhibiting cell proliferation (by MTT assay), MMPs secretion (by gelatinase zymography), cell invasion (through Matrigel) and induction of apoptosis (by live green caspase). In addition, NM was shown to down-regulate urokinase plasminogen activator (by fibrin zymography) and up-regulate tissue inhibitors of metalloproteinases (by reverse zymography) in another HCC cell line, SK-Hep-1. MMP-2 and MMP-9 activities were further modulated by phorbol 12-myristate 13-acetate (PMA) induction and inhibited by NM. In previous studies, NM inhibited Sk-Hep-1 xenografts in nude mice and also inhibited hepatic metastasis of B16FO melanoma cells. Our results suggest that NM is an excellent candidate for therapeutic use in the treatment HCC by inhibiting critical parameters in cancer development and progression, such as proliferation, invasion and metastasis, and by inducing apoptosis.

Markers of endothelial dysfunction and severity of hypoxaemia in the Eisenmenger syndrome.

PubMed

de P S Soares, Rosangela; Maeda, Nair Y; Bydlowski, Sérgio P; Lopes, Antonio Augusto

2005-10-01

Endothelial dysfunction has been reported in hypoxaemic patients with the Eisenmenger syndrome, but a direct correlation between levels of endothelial markers and the severity of hypoxaemia has not been explored. With this in mind, we compared the levels in the plasma of tissue-type plasminogen activator, thrombomodulin, and von Willebrand factor in 25 patients with the Eisenmenger syndrome. They had a median age of 31 years, and were divided into 2 groups according to their recent clinical history. Thus, 18 patients were stable, being in functional class II or III, seen as outpatients, and having peripheral saturations of oxygen of 89 plus or minus 5 percent. In contrast, 7 patients were unstable, showing episodes of symptoms placing them in functional class IV, requiring care in hospital, and manifesting saturations of oxygen of 77 plus or minus 5 percent. We were able to follow 12 patients, 8 who were stable and 4 unstable, for 24 months. At baseline, levels of von Willebrand factor were higher in the unstable patients when compared to those who were stable, at 142 plus or minus 29 and 110 plus or minus 25 units per decilitre, respectively (p equal to 0.013). This correlated positively with oxygen desaturation (p less than 0.020). The structural abnormalities also correlated positively with the magnitude of hypoxaemia (p less than 0.020). Levels remained higher in the unstable patients throughout the period of follow-up (p equal to 0.006). Tissue-type plasminogen activator was also increased, at 14.3 plus or minus 8.4 versus 6.5 plus or minus 2.7 nanograms per millilitre in controls (p less than 0.001), whereas thrombomodulin was decreased, with values of 14.4 versus 34.6 nanograms per millilitre in controls (p for median values of less than 0.001). There was no correlation with saturations of oxygen. We conclude that measurement of von Willebrand factor, as compared with tissue-type plasminogen activator and thrombomodulin, will prove a better marker of endothelial response to hypoxaemia in patients with the Eisenmenger syndrome.

Plasminogen activator inhibitor 1 is elevated in the children of men with premature myocardial infarction.

PubMed

Rallidis, L S; Megalou, A A; Papageorgakis, N H; Trikas, A G; Chatzidimitriou, G I; Tsitouris, G K

1996-09-01

To assess whether plasminogen activator inhibitor 1 (PAI-1) activity is elevated in the progeny of young coronary men, 193 young subjects were recruited and divided into two groups. Group A consisted of 104 children whose fathers had suffered a myocardial infarction before the age of 55 ("cases"). Eighty-nine young subjects matched for age, sex, body mass index (BMI) and smoking habits without familial history of coronary artery disease (CAD) served as controls (group B). Children with a family history of diabetes mellitus or hypertension were excluded from both groups. We measured PAI-1 activity, tissue-type plasminogen activator (t-PA) antigen, a2-antiplasmin, fibrinogen, lipids and apolipoproteins in both groups. PAI-1 activity levels were also determined in the men who suffered a premature myocardial infarction 4 months after their discharge. PAI-1 activity levels were higher in cases compared to controls (3.13 +/- 1.9 vs 2.17 +/- 1.9 U/ml, p = 0.0014). t-PA antigen and a2-antiplasmin did not differ significantly between the two groups, while fibrinogen, total cholesterol, low-density lipoprotein cholesterol, apolipoprotein B and lipoprotein(a) were significantly higher in group A. PAI-1 was positively correlated with triglycerides (r = 0.22, p = 0.024), apolipoprotein B (r = 0.21, p = 0.039) and fibrinogen (r = 0.22, p = 0.029) in cases and with BMI in both cases (r = 0.37, p = 0.0003) and controls (r = 0.23, p = 0.044). In stepwise multiple regression analysis, only apolipoprotein B (p = 0.008) and BMI (p = 0.0014) were significant determinants of PAI-1 activity in cases. There was also a positive correlation between PAI-1 activity levels of the affected fathers and their children (r = 0.30, p = 0.01). The present data support the hypothesis that elevated PAI-1 levels in the offspring of men with premature myocardial infarction impair their fibrinolytic capacity contributing to their familial predisposition to CAD.

Circadian variability of fibrinolytic markers and endothelial function in patients with obstructive sleep apnea.

PubMed

Bagai, Kanika; Muldowney, James A S; Song, Yanna; Wang, Lily; Bagai, Jayant; Artibee, Kay J; Vaughan, Douglas E; Malow, Beth A

2014-02-01

Obstructive sleep apnea (OSA) is strongly associated with cardiovascular disease, including stroke and acute coronary syndromes. Plasminogen activator inhibitor-1 (PAI-1), the principal inhibitor of tissue-type plasminogen activator (t-PA), has a pronounced circadian rhythm and is elevated in both OSA and cardiovascular disease and may be an important link between the two conditions. Endothelial dysfunction is one of the underlying pathophysiological mechanisms of cardiovascular disease, and may be altered in OSA. Our primary aim was to compare circadian variability of PAI-1 and t-PA in patients with OSA and normal controls by determining the amplitude (peak level) and mesor (rhythm adjusted mean) of PAI-1 and t-PA in serial blood samples over a 24-h period. The secondary aim was to measure markers of endothelial function (brachial and radial artery flow) in patients with OSA compared with normal controls. Cross-sectional cohort study. Subjects age 18 y or older, with a body mass index of 25-45 kg/m(2), with or without evidence of untreated OSA. Plasma samples were collected every 2 h, in OSA patients and matched controls, over a 24-h period. PAI-1 and t-PA antigen and activity were measured. The presence or absence of OSA (apnea-hypopnea index of 5 or greater) was confirmed by overnight polysomnography. Endothelial function was measured via brachial artery flow mediated vasodilatation and computerized arterial pulse waveform analysis. The rhythm-adjusted mean levels of PAI-1 antigen levels in the OSA group (21.8 ng/mL, 95% confidence level [CI], 18 to 25.7) were significantly higher as compared to the non-OSA group (16 ng/mL, 95% CI, 12.2 to 19.8; P = 0.03). The rhythm-adjusted mean levels of PAI-1 activity levels in the OSA group (23.9 IU/mL, 95% CI, 21.4 to 26.5) were also significantly higher than in the non-OSA group (17.2 IU/ mL, 95% CI, 14.6 to 19.9; P < 0.001).There were strong correlations between amplitude of PAI-1 activity and severity of OSA as measured by AHI (P = 0.02), and minimum oxygen levels during sleep (P = 0.04). Endothelial function parameters did not differ significantly between the two groups. The presence of obstructive sleep apnea adversely affects circadian fibrinolytic balance with higher mean plasminogen activator inhibitor-1 activity and antigen, and significantly lower mean tissue-type plasminogen activator activity compared with controls. This perturbation may be an important mechanism for increased cardiovascular events in patients with obstructive sleep apnea. Intermittent hypoxia and changes in circadian clock gene activity in obstructive sleep apnea may be responsible for these findings and warrant further study. Favorable changes in fibrinolytic balance may underlie the reduction in cardiovascular events observed with the treatment of obstructive sleep apnea.

Potency and stability of frozen urokinase solutions in syringes.

PubMed

Dedrick, Stephen C; Ramirez-Rico, José

2004-08-01

The stability and potency of frozen urokinase solutions in syringes were studied. To determine the stability and potency of compounded urokinase dilutions after multiple freeze-thaw cycles, a total of 160 syringes containing five urokinase concentrations (2,500, 5,000, 7,500, 12,500, and 25,000 IU/mL) were prepared. For each of the five concentrations tested, two syringes per concentration were reserved for baseline testing. The remaining 150 syringes were frozen at -30 degrees C. After 7 days, half of the syringes (group 1) were thawed at room temperature, tested, and left at room temperature for 12 hours before refreezing. The other half of the syringes (group 2) were kept frozen for 30 days. Thirty days after initial compounding, all syringes were thawed, and the samples' urokinase potency, pH, and physical appearance were evaluated. Syringes were visually inspected for color, clarity, and precipitation. Descriptive statistics were computed for each concentration group and testing day. The compounded dilutions were stable under each experimental condition, with no physical deterioration or loss of in vitro potency after two freeze-thaw cycles. The reduced waste associated with the ability to refreeze unused urokinase could substantially lower the cost of procedures such as thrombolysis after intraventricular hemorrhage and catheter clearance by as much as 95%. Dilutions of urokinase 2,500-25,000 IU/mL were stable in single-use syringes after being frozen for 7 days, thawed, and refrozen for another 23 days.

Decreased levels of active uPA and KLK8 assessed by [111 In]MICA-401 binding correlate with the seizure burden in an animal model of temporal lobe epilepsy.

PubMed

Missault, Stephan; Peeters, Lore; Amhaoul, Halima; Thomae, David; Van Eetveldt, Annemie; Favier, Barbara; Thakur, Anagha; Van Soom, Jeroen; Pitkänen, Asla; Augustyns, Koen; Joossens, Jurgen; Staelens, Steven; Dedeurwaerdere, Stefanie

2017-09-01

Urokinase-type plasminogen activator (uPA) and kallikrein-related peptidase 8 (KLK8) are serine proteases that contribute to extracellular matrix (ECM) remodeling after brain injury. They can be labelled with the novel radiotracer [ 111 In]MICA-401. As the first step in exploring the applicability of [ 111 In]MICA-401 in tracing the mechanisms of postinjury ECM reorganization in vivo, we performed in vitro and ex vivo studies, assessing [ 111 In]MICA-401 distribution in the brain in two animal models: kainic acid-induced status epilepticus (KASE) and controlled cortical impact (CCI)-induced traumatic brain injury (TBI). In the KASE model, in vitro autoradiography with [ 111 In]MICA-401 was performed at 7 days and 12 weeks post-SE. To assess seizure burden, rats were monitored using video-electroencephalography (EEG) for 1 month before the 12-week time point. In the CCI model, in vitro autoradiography was performed at 4 days and ex vivo autoradiography at 7 days post-TBI. At 7 days post-SE, in vitro autoradiography revealed significantly decreased [ 111 In]MICA-401 binding in hippocampal CA3 subfield and extrahippocampal temporal lobe (ETL). In the chronic phase, when animals had developed spontaneous seizures, specific binding was decreased in CA3 and CA1/CA2 subfields of hippocampus, dentate gyrus, ETL, and parietal cortex. Of interest, KASE rats with the highest frequency of seizures had the lowest hippocampal [ 111 In]MICA-401 binding (r = -0.76, p ≤ 0.05). Similarly, at 4 days post-TBI, in vitro [ 111 In]MICA-401 binding was significantly decreased in medial and lateral perilesional cortex and ipsilateral dentate gyrus. Ex vivo autoradiography at 7 days post-TBI, however, revealed increased tracer uptake in perilesional cortex and hippocampus, which was likely related to tracer leakage due to blood-brain barrier (BBB) disruption. Strong association of reduced [ 111 In]MICA-401 binding with seizure burden in the KASE model suggests that analysis of reduced levels of active uPA/KLK8 represents a novel biomarker candidate to be explored as a biomarker for epilepsy severity. However, limited BBB permeability of [ 111 In]MICA-401 currently limits its application in vivo. Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.

High-fat diet enhances and plasminogen activator inhibitor-1 deficiency attenuates bone loss in mice with Lewis Lung carcinoma

USDA-ARS?s Scientific Manuscript database

This study determined the effects of a high-fat diet and plasminogen activator inhibitor-1 deficiency (PAI-1-/-) on bone structure in mice bearing Lewis lung carcinoma (LLC) in lungs. Reduction in bone volume fraction (BV/TV) by 22% and 21%, trabecular number (Tb.N) by 8% and 4% and bone mineral de...

Plasminogen fragments K 1-3 and K 5 bind to different sites in fibrin fragment DD.

PubMed

Grinenko, T V; Kapustianenko, L G; Yatsenko, T A; Yusova, O I; Rybachuk, V N

2016-01-01

Specific plasminogen-binding sites of fibrin molecule are located in Аα148-160 regions of C-terminal domains. Plasminogen interaction with these sites initiates the activation process of proenzyme and subsequent fibrin lysis. In this study we investigated the binding of plasminogen fragments K 1-3 and K 5 with fibrin fragment DD and their effect on Glu-plasminogen interaction with DD. It was shown that the level of Glu-plasminogen binding to fibrin fragment DD is decreased by 50-60% in the presence of K 1-3 and K 5. Fragments K 1-3 and K 5 have high affinity to fibrin fragment DD (Kd is 0.02 for K 1-3 and 0.054 μМ for K 5). K 5 interaction is independent and K 1-3 is partly dependent on C-terminal lysine residues. K 1-3 interacts with complex of fragment DD-immobilized K 5 as well as K 5 with complex of fragment DD-immobilized K 1-3. The plasminogen fragments do not displace each other from binding sites located in fibrin fragment DD, but can compete for the interaction. The results indicate that fibrin fragment DD contains different binding sites for plasminogen kringle fragments K 1-3 and K 5, which can be located close to each other. The role of amino acid residues of fibrin molecule Аα148-160 region in interaction with fragments K 1-3 and K 5 is discussed.

Administration of antioxidant vitamins does not alter plasma fibrinolytic activity in subjects with central obesity.

PubMed

Rifici, V A; Schneider, S H; Chen, Y; Khachadurian, A K

1997-09-01

In vitro studies suggest that oxidized low density lipoprotein inhibits fibrinolysis by stimulating the production of plasminogen activator inhibitor -1 (PAI). We assessed the effects of dietary antioxidant vitamins for four weeks on three indices of copper mediated oxidation of very low and low density lipoproteins (VLDL+LDL) and plasma fibrinolytic activities in 15 male subjects with central obesity, a condition associated with increased PAI activity. Vitamin administration resulted in a decrease in production of thiobarbituric acid reactive substances from 29.3 +/- 3.9 to 13.6 +/- 3.5 nmoles/mg VLDL + LDL protein (mean +/- SE, p <0.003), an increase in the lag phase of conjugated diene formation from 94.8 +/- 5.5 to 225.0 +/- 31.9 min (p <0.001) and an increase in reactivity of lysine residues from 73.6% +/- 4.8% to 86.8% +/- 3.6% (p <0.034) demonstrating a reduction in the susceptibility of the lipoproteins to oxidation. However, antioxidant vitamins had no effect on plasma PAI activity, PAI antigen, tissue-type plasminogen activator activity and antigen, fibrinogen and fibrin degradation products. These results do not support the hypothesis that lipoprotein oxidation is a significant cause of impaired fibrinolysis in men with central obesity.

Joint analysis of multiple biomarkers for identifying type 2 diabetes in middle-aged and older Chinese: a cross-sectional study

PubMed Central

Wu, Hongyu; Yu, Zhijie; Qi, Qibin; Li, Huaixing; Sun, Qi

2011-01-01

Objective Identifying individuals with high risk of type 2 diabetes is important. To evaluate discriminatory ability of multiple biomarkers for type 2 diabetes in a Chinese population. Methods Plasma adiponectin, plasminogen activator inhibitor-1, retinol-binding protein 4, resistin, C-reactive protein, interleukin 6 (IL-6), tumour necrosis factor α receptor 2 and ferritin were measured in a population-based sample of 3189 Chinese (1419 men and 1770 women) aged 50–70 years. A weighted biomarkers risk score (BRS) was developed based on the strength of associations of these biomarkers with type 2 diabetes. The discriminatory ability was tested by the area under receiver operating characteristics curve (AUC). Results Adiponectin, plasminogen activator inhibitor-1, IL-6 and ferritin were independently associated with the prevalence of type 2 diabetes, and they were used to calculate the biomarkers risk score (BRS). After adjustment for the confounding factors, the ORs for type 2 diabetes and impaired fasting glucose with each point increment of BRS were 1.28 (95% CI 1.22 to 1.34) and 1.16 (1.12 to 1.20), respectively. Compared with those in the lowest quintile of the BRS, the participants in the highest quintile have an OR (95% CI) of 6.67 (4.21 to 10.55) for type 2 diabetes. The area under the curve for the BRS and conventional risk factors alone was 0.73 and 0.76, respectively, and substantially increased to 0.81 after combining both BRS and conventional risk factors (p<0.001). Conclusions These data suggest that combining multiple biomarkers and conventional risk factors might substantially enhance the ability to identify individuals with type 2 diabetes. More prospective data are warranted to confirm this observation. PMID:22021786

Phosphatidylserine as an anchor for plasminogen and its plasminogen receptor, Histone H2B, to the macrophage surface

PubMed Central

DAS, R.; PLOW, E. F.

2013-01-01

Summary Background Plasminogen (Plg) binding to cell surface Plg receptors (Plg-Rs) on the surface of macrophages facilitates Plg activation and migration of these cells. Histone H2B (H2B) acts as a Plg-R and its cell surface expression is upregulated when monocytes are differentiated to macrophages via a pathway dependent on L-type Ca2+ channels and intracellular Ca2+. Objectives We sought to investigate the mechanism by which H2B, a protein without a transmembrane domain, is retained on themacrophage surface. Methods THP-1 monocytoid cells were induced to differentiate with interferon gamma + Vitamin D3 or to undergo apoptosis by treatment with camptothecin. Flow cytometry and cell surface biotinylation followed by Western blotting were used to measure the interrelationship between Plg binding, cell surface expression of H2B and outermembrane exposure of phosphatidylserine (PS). Results H2B interacted directly with PS via an electrostatic interaction. Anti-PS or PS binding proteins, annexin V and protein S, diminished H2B interaction with PS on the surface of differentiated or apoptotic cells and these same reagents inhibited Plg binding to these cells. L-type Ca2+ channels played a significant role in PS exposure, H2B surface expression and Plg binding induced either by differentiation or apoptosis. Conclusions These data suggest that H2B tethers to the surface of cells by interacting with PS on differentiated or apoptotic monocytoid cells. L-type Ca2+ channels regulate PS exposure on the surface of these cells. The exposed PS interacts directly with H2B and hence provides sites for Plg to bind to. PMID:21040449

Dual-targeting Wnt and uPA receptors using peptide conjugated ultra-small nanoparticle drug carriers inhibited cancer stem-cell phenotype in chemo-resistant breast cancer.

PubMed

Miller-Kleinhenz, Jasmine; Guo, Xiangxue; Qian, Weiping; Zhou, Hongyu; Bozeman, Erica N; Zhu, Lei; Ji, Xin; Wang, Y Andrew; Styblo, Toncred; O'Regan, Ruth; Mao, Hui; Yang, Lily

2018-01-01

Heterogeneous tumor cells, high incidence of tumor recurrence, and decrease in overall survival are the major challenges for the treatment of chemo-resistant breast cancer. Results of our study showed differential chemotherapeutic responses among breast cancer patient derived xenograft (PDX) tumors established from the same patients. All doxorubicin (Dox)-resistant tumors expressed higher levels of cancer stem-like cell biomarkers, including CD44, Wnt and its receptor LRP5/6, relative to Dox-sensitive tumors. To effectively treat resistant tumors, we developed an ultra-small magnetic iron oxide nanoparticle (IONP) drug carrier conjugated with peptides that are dually targeted to Wnt/LRP5/6 and urokinase plasminogen activator receptor (uPAR). Our results showed that simultaneous binding to LRP5/6 and uPAR by the dual receptor targeted IONPs was required to inhibit breast cancer cell invasion. Molecular analysis revealed that the dual receptor targeted IONPs significantly inhibited Wnt/β-catenin signaling and cancer stem-like phenotype of tumor cells, with marked reduction of Wnt ligand, CD44 and uPAR. Systemic administration of the dual targeted IONPs led to nanoparticle-drug delivery into PDX tumors, resulting in stronger tumor growth inhibition compared to non-targeted or single-targeted IONP-Dox in a human breast cancer PDX model. Therefore, co-targeting Wnt/LRP and uPAR using IONP drug carriers is a promising therapeutic approach for effective drug delivery to chemo-resistant breast cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

Targeted thrombolysis by using of magnetic mesoporous silica nanoparticles.

PubMed

Wang, Mingqi; Zhang, Jixi; Yuan, Ziming; Yang, Wenzhi; Wu, Qiang; Gu, Hongchen

2012-08-01

Thrombolytics inevitably led to the risk of hemorrhagic complications due to their non-specific plasminogen activation in treatment of thrombosis. The aim of this study was to determine whether a kind of superparamagnetic mesoporous silica nanoparticle with expanded pore size could achieve effectively targeted thrombolysis. The magnetic mesoporous silica nanoparticles (M-MSNs) with the pore size of 6 nm were prepared by method of the surfactant templating on nano magnetic particles. We investigated the feasibility and efficacy of target thrombolysis with the resultant spheres through fibrin agarose plate assay (FAPA) and a dynamic flow system in vitro. It displayed a 30-fold enhancement of urokinase (UK) loading capacity over the particles without mesoporous layer or the magnetic spheres with mesopores of 3.7 nm. A sustained release behavior was observed due to its larger pore size, higher surface area and narrow mesopore channals contrast to non-mesoporous and small mesopore of 3.7 nm controls. Meanwhile, fibrin agarose plate assay revealed that UK/M-MSNs exhibited a more rapid growth rate of thrombolysis even lasting for 3 days. Additionally, flow model test in vitro suggested this kind of nanoparticle complex enhanced the thrombolysis efficacy by 3.5 fold over the same amount of native UK in 30 min. When compared to non-mesoporous and small mesopore controls, it also represented an extremely higher lysis efficiency (ANOVA, P < 0.01) and a shorter reperfusion time (ANOVA, P < 0.001). Such a magnetic mesoporous silica nanoparticle carrier was expected to be further studied for targeted thrombolytic therapy.

Combinatorial gene therapy renders increased survival in cirrhotic rats

PubMed Central

2010-01-01

Background Liver fibrosis ranks as the second cause of death in México's productive-age population. This pathology is characterized by acummulation of fibrillar proteins in hepatic parenchyma causing synthetic and metabolic disfunction. Remotion of excessive fibrous proteins might result in benefit for subjects increasing survival index. The goal of this work was to find whether the already known therapeutical effect of human urokinase Plasminogen Activator and human Matrix Metalloprotease 8 extends survival index in cirrhotic animals. Methods Wistar rats (80 g) underwent chronic intoxication with CCl4: mineral oil for 8 weeks. Cirrhotic animals were injected with a combined dose of Ad-delta-huPA plus Ad-MMP8 (3 × 1011 and 1.5 × 1011 vp/Kg, respectively) or with Ad-beta-Gal (4.5 × 1011) and were killed after 2, 4, 6, 8 and 10 days. Then, liver and serum were collected. An additional set of cirrhotic animals injected with combined gene therapy was also monitored for their probability of survival. Results Only the cirrhotic animals treated with therapeutical genes (Ad-delta-huPA+Ad-MMP-8) showed improvement in liver fibrosis. These results correlated with hydroxyproline determinations. A significant decrement in alpha-SMA and TGF-beta1 gene expression was also observed. Cirrhotic rats treated with Ad-delta-huPA plus Ad-MMP8 had a higher probability of survival at 60 days with respect to Ad-beta-Gal-injected animals. Conclusion A single administration of Ad-delta-huPA plus Ad-MMP-8 is efficient to induce fibrosis regression and increase survival in experimental liver fibrosis. PMID:20509929

Pulmonary veins in the normal lung and pulmonary hypertension due to left heart disease

PubMed Central

Hunt, James M.; Bethea, Brian; Liu, Xiang; Gandjeva, Aneta; Mammen, Pradeep P. A.; Stacher, Elvira; Gandjeva, Marina R.; Parish, Elisabeth; Perez, Mario; Smith, Lynelle; Graham, Brian B.; Kuebler, Wolfgang M.

2013-01-01

Despite the importance of pulmonary veins in normal lung physiology and the pathobiology of pulmonary hypertension with left heart disease (PH-LHD), pulmonary veins remain largely understudied. Difficult to identify histologically, lung venous endothelium or smooth muscle cells display no unique characteristic functional and structural markers that distinguish them from pulmonary arteries. To address these challenges, we undertook a search for unique molecular markers in pulmonary veins. In addition, we addressed the expression pattern of a candidate molecular marker and analyzed the structural pattern of vascular remodeling of pulmonary veins in a rodent model of PH-LHD and in lung tissue of patients with PH-LHD obtained at time of placement on a left ventricular assist device. We detected urokinase plasminogen activator receptor (uPAR) expression preferentially in normal pulmonary veins of mice, rats, and human lungs. Expression of uPAR remained elevated in pulmonary veins of rats with PH-LHD; however, we also detected induction of uPAR expression in remodeled pulmonary arteries. These findings were validated in lungs of patients with PH-LHD. In selected patients with sequential lung biopsy at the time of removal of the left ventricular assist device, we present early data suggesting improvement in pulmonary hemodynamics and venous remodeling, indicating potential regression of venous remodeling in response to assist device treatment. Our data indicate that remodeling of pulmonary veins is an integral part of PH-LHD and that pulmonary veins share some key features present in remodeled yet not normotensive pulmonary arteries. PMID:24039255

Detection and characterization of a new metabolite of 17alpha-methyltestosterone.

PubMed

Pozo, Oscar J; Van Eenoo, Peter; Deventer, Koen; Lootens, Leen; Van Thuyne, Wim; Parr, Maria K; Schänzer, Wilhelm; Sancho, Juan V; Hernández, Felix; Meuleman, Philip; Leroux-Roels, Geert; Delbeke, Frans T

2009-11-01

The misuse of the anabolic steroid methyltestosterone is currently routinely monitored in doping control laboratories by gas chromatography-mass spectrometry (GC-MS) of two of its metabolites: 17alpha-methyl-5beta-androstane-3alpha,17beta-diol and 17alpha-methyl-5alpha-androstane-3alpha,17beta-diol. Because of the absence of any easy ionizable moiety, these metabolites are poorly detectable using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI). In this study, the metabolism of methyltestosterone has been reinvestigated by the use of a precursor ion scan method in LC-ESI-MS/MS. Two metabolites have been detected using this method. Both compounds have been confirmed in postadministration urine samples of an urokinase plasminogen activator-severe combined immunodeficiency (uPA-SCID) mouse with humanized liver and were characterized by LC-MS/MS and GC-MS using both quadrupole and time of flight analyzers. From the detailed study of the fragmentation, these metabolites were proposed to be epimethyltestosterone and a dehydrogenated compound. Epimethyltestosterone has previously been described as a minor metabolite, whereas the occurrence of the oxidized metabolite has not been reported. Comparison with the synthesized reference revealed that the structure of the dehydrogenated metabolite is 6-ene-epimethyltestosterone. A selected reaction monitoring method including three transitions for each metabolite has been developed and applied to samples from an excretion study and to samples declared positive after GC-MS analysis. 6-Ene-epimethyltestosterone was found in all samples, showing its applicability in the detection of methyltestosterone misuse.

Further analyses of human kidney cell populations separated on the Space Shuttle

NASA Technical Reports Server (NTRS)

Stewart, Robin M.; Todd, Paul; Cole, Kenneth D.; Morrison, Dennis R.

1992-01-01

Cultured human embryonic kidney cells were separated into electrophoretic subpopulations in laboratory experiments and in two separation experiments on the STS-8 (Challenger) Space Shuttle flight using the mid-deck Continuous Flow Electrophoretic Separator (CFES). Populations of cells from each fraction were cultured for the lifetime of the cells, and supernatant medium was withdrawn and replaced at 4-day intervals. Withdrawn medium was frozen at -120 C for subsequent analysis. Enzyme assays, antibodies and gel electrophoresis were used as analytical tools for the detection and quantization of plasminogen activators in these samples. These assays of frozen-culture supernatant fluids confirmed the electrophoretic separation of plasminogen-activator-producing cells from nonproducing cells, the isolation of cells capable of sustained production, and the separation of cells that produce different plasminogen activators from one other.

Tissue-type plasminogen activator-induced fibrinolysis is enhanced in patients with breast, lung, pancreas and colon cancer.

PubMed

Nielsen, Vance G; Matika, Ryan W; Ley, Michele L B; Waer, Amy L; Gharagozloo, Farid; Kim, Samuel; Nfonsam, Valentine N; Ong, Evan S; Jie, Tun; Warneke, James A; Steinbrenner, Evangelina B

2014-04-01

Although cancer-mediated changes in hemostatic proteins unquestionably promote hypercoagulation, the effects of neoplasia on fibrinolysis in the circulation are less well defined. The goals of the present investigation were to determine if plasma obtained from patients with breast, lung, pancreas and colon cancer was less or more susceptible to lysis by tissue-type plasminogen activator (tPA) compared to plasma obtained from normal individuals. Archived plasma obtained from patients with breast (n = 18), colon/pancreas (n = 27) or lung (n = 19) was compared to normal individual plasma (n = 30) using a thrombelastographic assay that assessed fibrinolytic vulnerability to exogenously added tPA. Plasma samples were activated with tissue factor/celite, had tPA added, and had data collected until clot lysis occurred. Additional, similar samples had potato carboxypeptidase inhibitor added to assess the role played by thrombin-activatable fibrinolysis inhibitor in cancer-modulated fibrinolysis. Rather than inflicting a hypofibrinolytic state, the three groups of cancers demonstrated increased vulnerability to tPA (e.g. decreased time to lysis, increased speed of lysis, decreased clot lysis time). However, hypercoagulation manifested as increased speed of clot formation and strength compensated for enhanced fibrinolytic vulnerability, resulting in a clot residence time that was not different from normal individual thrombi. In sum, enhanced hypercoagulability associated with cancer was in part diminished by enhanced fibrinolytic vulnerability to tPA.

VEGF correlates with inflammation and fibrosis in tuberculous pleural effusion.

PubMed

Bien, Mauo-Ying; Wu, Ming-Ping; Chen, Wei-Lin; Chung, Chi-Li

2015-01-01

To investigate the relationship among angiogenic cytokines, inflammatory markers, and fibrinolytic activity in tuberculous pleural effusion (TBPE) and their clinical importance. Forty-two patients diagnosed with TBPE were studied. Based on chest ultrasonography, there were 26 loculated and 16 nonloculated TBPE patients. The effusion size radiological scores and effusion vascular endothelial growth factor (VEGF), interleukin- (IL-) 8, plasminogen activator inhibitor type-1 (PAI-1), and tissue type plasminogen activator (tPA) were measured. Treatment outcome and pleural fibrosis, defined as radiological residual pleural thickening (RPT), were assessed at 6-month follow-up. The effusion size and effusion lactate dehydrogenase (LDH), VEGF, IL-8, PAI-1, and PAI-1/tPA ratio were significantly higher, while effusion glucose, pH value, and tPA were significantly lower, in loculated than in nonloculated TBPE. VEGF and IL-8 correlated positively with LDH and PAI-1/tPA ratio and negatively with tPA in both loculated and nonloculated TBPE. Patients with higher VEGF or greater effusion size were prone to develop RPT (n=14; VEGF, odds ratio 1.28, P=0.01; effusion size, odds ratio 1.01, P=0.02), and VEGF was an independent predictor of RPT in TBPE (receiver operating characteristic curve AUC=0.985, P<0.001). Effusion VEGF correlates with pleural inflammation and fibrosis and may be targeted for adjunct therapy for TBPE.

VEGF Correlates with Inflammation and Fibrosis in Tuberculous Pleural Effusion

PubMed Central

Bien, Mauo-Ying; Wu, Ming-Ping; Chen, Wei-Lin; Chung, Chi-Li

2015-01-01

Objective. To investigate the relationship among angiogenic cytokines, inflammatory markers, and fibrinolytic activity in tuberculous pleural effusion (TBPE) and their clinical importance. Methods. Forty-two patients diagnosed with TBPE were studied. Based on chest ultrasonography, there were 26 loculated and 16 nonloculated TBPE patients. The effusion size radiological scores and effusion vascular endothelial growth factor (VEGF), interleukin- (IL-) 8, plasminogen activator inhibitor type-1 (PAI-1), and tissue type plasminogen activator (tPA) were measured. Treatment outcome and pleural fibrosis, defined as radiological residual pleural thickening (RPT), were assessed at 6-month follow-up. Results. The effusion size and effusion lactate dehydrogenase (LDH), VEGF, IL-8, PAI-1, and PAI-1/tPA ratio were significantly higher, while effusion glucose, pH value, and tPA were significantly lower, in loculated than in nonloculated TBPE. VEGF and IL-8 correlated positively with LDH and PAI-1/tPA ratio and negatively with tPA in both loculated and nonloculated TBPE. Patients with higher VEGF or greater effusion size were prone to develop RPT (n = 14; VEGF, odds ratio 1.28, P = 0.01; effusion size, odds ratio 1.01, P = 0.02), and VEGF was an independent predictor of RPT in TBPE (receiver operating characteristic curve AUC = 0.985, P < 0.001). Conclusions. Effusion VEGF correlates with pleural inflammation and fibrosis and may be targeted for adjunct therapy for TBPE. PMID:25884029

Clustering of haemostatic variables and the effect of high cashew and walnut diets on these variables in metabolic syndrome patients.

PubMed

Pieters, Marlien; Oosthuizen, Welma; Jerling, Johann C; Loots, Du Toit; Mukuddem-Petersen, Janine; Hanekom, Susanna M

2005-09-01

We investigated the effect of a high walnut and cashew diet on haemostatic variables in people with the metabolic syndrome. Factor analysis was used to determine how the haemostatic variables cluster with other components of the metabolic syndrome and multiple regression to determine possible predictors. This randomized, control, parallel, controlled-feeding trial included 68 subjects who complied with the Third National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol criteria. After a 3-week run-in following the control diet, subjects were divided into three groups receiving either walnuts or cashews (20 energy%) or a control diet for 8 weeks. The nut intervention had no significant effect on von Willebrand factor antigen, fibrinogen, factor VII coagulant activity, plasminogen activator inhibitor 1 activity, tissue plasminogen activator activity or thrombin activatable fibrinolysis inhibitor. Statistically, fibrinogen clustered with the body-mass-correlates and acute phase response factors, and factor VII coagulant activity clustered with high-density lipoprotein cholesterol (HDL-C). Tissue plasminogen activator activity, plasminogen activator inhibitor 1 activity and von Willebrand factor antigen clustered into a separate endothelial function factor. HDL-C and markers of obesity were the strongest predictors of the haemostatic variables. We conclude that high walnut and cashew diets did not influence haemostatic factors in this group of metabolic syndrome subjects. The HDL-C increase and weight loss may be the main focus of dietary intervention for the metabolic syndrome. Furthermore, diet composition may have only limited effects if weight loss is not achieved.