Effects of hypoxia and reoxygenation on the expression levels of the urokinase-type plasminogen activator, its inhibitor plasminogen activator inhibitor type-1 and the urokinase-type plasminogen activator receptor in human head and neck tumour cells.

PubMed

Sprague, Lisa D; Tomaso, Herbert; Mengele, Karin; Schilling, Daniela; Bayer, Christine; Stadler, Peter; Schmitt, Manfred; Molls, Michael

2007-05-01

One aim during oncological radiation therapy is to induce reoxygenation in hypoxic tumours in order to enhance radiosensitivity and ultimately increase cell death. In squamous cell carcinomas of the head and neck (SCCHN), hypoxia is considered a pivotal physiological modulator for malignant progression, whereby the plasminogen activation system is involved in overlapping functions such as the shaping of the extracellular matrix, cell proliferation and signal transduction. Since little is known about reoxygenation and the plasminogen activation system in SCCHN, three human SCCHN cell lines (BHY, FaDu, and CAL27) and a non-transformed control cell line (VH7) were exposed to hypoxic (<0.5% O2) conditions for up to 72 h and subsequently reoxygenated for 24 h at normoxic conditions. The mRNA expression of the urokinase-type plasminogen activator (uPA), the plasminogen activator inhibitor type-1 (PAI-1) and the urokinase-type plasminogen activator receptor (uPAR) was assessed by means of real-time semi-quantitative RT-PCR, and the protein expression was determined by immunoenzymometric quantification (ELISA). Both hypoxia and reoxygenation induced statistically significant changes in uPA, PAI-1 and uPAR mRNA and protein levels in the various cell lines investigated, showing that oxygen tension is a strong modulator of the plasminogen activation system in vitro. However, no uniform correlation pattern was found between the mRNA and protein levels analysed over all three time-points (24, 48, and 72 h) and oxygen treatment variants (N, H, R) nor according to oxygen treatment conditions over all three time-points. Changes in oxygen tension could therefore be modulating the fragile balance between the various components of the plasminogen activation system in SSCHN ultimately leading to an increased tumour matrix disruption, alterations in cell invasiveness, and the dissemination of tumour cells to distant organs.

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.

Urokinase injection-triggered clearance enhancement of a 4-arm PEG-conjugated 64Cu-bombesin analog tetramer: A novel approach for the improvement of PET imaging contrast.

PubMed

Matsumura, Kazushi; Zouda, Maki; Wada, Yasuhiro; Yamashita, Fumiyoshi; Hashida, Mitsuru; Watanabe, Yasuyoshi; Mukai, Hidefumi

2018-05-07

Radiolabeled antibodies, polyethylene glycol-conjugated (PEGylated) peptides, liposomes, and other materials were investigated as positron-emission tomography (PET) probes. These substances accumulate in tumors but often remain too long in circulation. We investigated the combination of intravenous urokinase injection and its substrate linker as a triggered radioisotope clearance enhancement system to improve imaging contrast. To this end, we synthesized a four-arm PEGylated 64 Cu-bombesin analog tetramer with a urokinase substrate linker. In mouse blood, it was almost perfectly cleaved and degraded into smaller radioactive fragments in vitro with urokinase (≥20,000 IU/mL). In mouse blood circulation, ∼50-65% of the probe was rapidly degraded after the urokinase injection and the radioactive fragments were eliminated mainly from the kidney. In contrast, tumor radioactivity levels did not change, and therefore, the tumors were clearly visualized. The tumor/blood ratio, an indicator of imaging contrast, increased 2.5 times, while elimination of the radioisotope from the blood was enhanced. This approach has the potential to improve imaging contrast using various PET probes. It could also shorten the time required to obtain sufficient contrast and decrease patient radiation exposure. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Measuring the metastatic potential of cancer cells

NASA Technical Reports Server (NTRS)

Morrison, Dennis R.; Gratzner, Howard; Atassi, M. Z.

1993-01-01

Cancer cells must secrete proteolytic enzymes to invade adjacent tissues and migrate to a new metastatic site. Urokinase (uPA) is a key enzyme related to metastasis in cancers of the lung, colon, gastric, uterine, breast, brain, and malignant melanoma. A NASA technology utilization project has combined fluorescence microscopy, image analysis, and flow cytometry, using fluorescent dyes, and urokinase-specific antibodies to measure uPA and abnormal DNA levels (related to cancer cell proliferation) inside the cancer cells. The project is focused on developing quantitative measurements to determine if a patient's tumor cells are actively metastasizing. If a significant number of tumor cells contain large amounts of uPA (esp. membrane-bound) then the post-surgical chemotherapy or radiotherapy can be targeted for metastatic cells that have already left the primary tumor. These analytical methods have been applied to a retrospective study of biopsy tissues from 150 node negative, stage 1 breast cancer patients. Cytopathology and image analysis has shown that uPA is present in high levels in many breast cancer cells, but not found in normal breast. Significant amounts of uPA also have been measured in glioma cell lines cultured from brain tumors. Commercial applications include new diagnostic tests for metastatic cells, in different cancers, which are being developed with a company that provides a medical testing service using flow cytometry for DNA analysis and hormone receptors on tumor cells from patient biopsies. This research also may provide the basis for developing a new 'magic bullet' treatment against metastasis using chemotherapeutic drugs or radioisotopes attached to urokinase-specific monoclonal antibodies that will only bind to metastatic cells.

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

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

Modulation of the malignant phenotype with the urokinase-type plasminogen activator and the type I plasminogen activator inhibitor.

PubMed

Sordat, B; Reiter, L; Cajot, J F

1990-12-02

Gene transfer techniques were utilized to evaluate the role of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in enhancing or preventing the expression of the invasive malignant phenotype, respectively. Mouse L-cell transfectants expressing human uPA or human PAI-1 as well as mouse B16 transfectants expressing mouse uPA or human PAI-1 were generated. These transfectants were tested using a variety of experimental methods including smooth muscle cell matrix solubilization in vitro, lung colony formation in vivo and co-cultures of antagonist-expressing cells in vitro. Results from these studies provide direct evidence for an enhancing role of uPA in malignant invasion and experimental metastasis and for a modulatory role of PAI-1 in tumor cell-mediated breakdown of extracellular matrices.

Structural Basis of Interaction between Urokinase-Type Plasminogen Activator and Its Receptor

PubMed Central

Barinka, Cyril; Parry, Graham; Callahan, Jennifer; Shaw, David E.; Kuo, Alice; Bdeir, Khalil; Cines, Douglas B.; Mazar, Andrew; Lubkowski, Jacek

2009-01-01

Summary Recent studies indicate that binding of urokinase-type plasminogen activator (uPA) to its high affinity receptor (uPAR), orchestrates uPAR interactions with other cellular components that play a pivotal role in diverse (patho-)physiological processes including wound healing, angiogenesis, inflammation, and cancer metastasis. However, notwithstanding the wealth of biochemical data available describing the activities of uPAR, little is known as to the exact mode of uPAR-uPA interactions and the presumed conformational changes that accompanying uPA-uPAR engagement. Here we report the crystal structure of soluble urokinase plasminogen activator receptor (suPAR), which contains the three domains of the wild-type receptor but lacks the cell surface anchoring sequence, in complex with the amino terminal fragment of urokinase-type plasminogen activator (ATF), at the resolution of 2.8 Å. We also report the 1.9 Å crystal structure of the free ATF. Our results provide a structural basis, represented by conformational changes induced in uPAR, for several published biochemical observations describing the nature of uPAR-uPA interactions and provide insight into mechanisms that may be responsible for the cellular responses induced by uPA binding. PMID:16979660

Electrophoretic separation of human kidney cells at zero gravity

NASA Technical Reports Server (NTRS)

Barlow, G. H.; Lazer, S. L.; Rueter, A.; Allen, R. E.

1977-01-01

Electrophoretic isolation of cells results in a loss of resolution power caused by the sedimentation of the cells in the media. The results of an experiment to extract urokinase from human embryos during the Apollo Soyuz mission are presented and discussed.

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

Colonic epithelial cell activation and the paradoxical effects of butyrate.

PubMed

Gibson, P R; Rosella, O; Wilson, A J; Mariadason, J M; Rickard, K; Byron, K; Barkla, D H

1999-04-01

Butyrate may have paradoxical effects on epithelial cells of similar origin. This study aimed to examine the hypothesis that one mechanism that dictates a cell's response to butyrate is its state of activation. First, the responses to 24 h exposure to butyrate (1-2 mM) of normal and neoplastic human colonic epithelial cells activated by their isolation and primary culture, and of colon cancer cell lines, LIM1215 and Caco-2, were examined. In primary cultures of normal and cancer cells, butyrate had no effect on alkaline phosphatase activities but significantly suppressed urokinase receptor expression by a mean +/- SEM of 30 +/- 12% and 36 +/- 9%, respectively. Interleukin-8 secretion was suppressed by 44 +/- 7% in normal cells (P < 0.05) but was unchanged in cancer cells. In contrast, the cell lines significantly increased alkaline phosphatase activities by >50%, urokinase receptor expression >2-fold and interleukin-8 secretion >3-fold in response to butyrate. Secondly, the effect of butyrate on Caco-2 cells was examined with or without prior exposure to a specific activating stimulus [tumour necrosis factor alpha (TNF alpha)]. Interleukin-8 secretion increased by 145 +/- 23% and 132 +/- 17% on 24 h exposure to 2 mM butyrate or 0.1 microM TNF alpha alone, respectively. However, in cells pre-treated with TNF alpha, butyrate significantly inhibited secretion by 34 +/- 7% below unstimulated levels. The response to butyrate of urokinase receptor, whose expression was not stimulated by TNF alpha, was unchanged. These effects were mimicked by trichostatin A, an inhibitor of histone deacetylase, suggesting that butyrate's paradoxical effects may have been operating by the same mechanism. In conclusion, some of the paradoxical effects of butyrate do not appear to represent inherent differences between normal and transformed cells. Rather, the response may be determined by the state of activation of the cells.

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.

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

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 effects of blood pressure and urokinase on brain injuries after experimental cerebral infarction in rats.

PubMed

Ji, Xunming; Li, Ke; Li, Wenbin; Li, Shuting; Yan, Feng; Gong, Wei; Luo, Yumin

2009-03-01

With the proposal of penumbra theory and development of intra-arterial thrombolysis (such as urokinase), the outcome of ischemic cerebrovascular disease is greatly improved. However, the incidence of hemorrhagic transformation (HT) increased concomitantly, and some studies showed a close relationship between blood pressure and HT. The mechanisms of blood pressure and urokinase effect on the incidence of HT are not clear. In this study, we investigated the effects of the different levels of blood pressure and urokinase on the ischemic lesions, the incidence of HT and the expression of matrix metalloproteinase 9 (MMP-9) in the rat ischemia-reperfusion models. Temporary focal ischemia was induced in male Sprague-Dawley rats using the intraluminal vascular occlusion method. The animals were assigned into four groups (n=11 in each group): low blood pressure group (LP), normal blood pressure group (NP), high blood pressure group (HP) and urokinase/high blood pressure group (UKHP). Adnephrin was applied to enhance the mean arterial blood pressure (MABP) at the beginning of reperfusion. MABP was maintained 20 mmHg higher than the baseline for 1 hour. Sodium nitroprusside was used to decrease MABP by 20 mmHg lower than the baseline for 1 hour. Both urokinase and adnephrin were used concomitantly in the UKHP group. Neurological deficit scores were evaluated at 2 hours (R2h) and 24 hours (R24h) after reperfusion. All rats were decapitated, their brains were sliced into 15-mum-thick slices, and the infarct volume and the visible HT were analysed. Three rats in each group were taken for immunohistochemistry and pathological analysis. There was no significant difference in MABP among the groups at the baseline time points (p>0.05), but blood pressure are definitely increased and decreased in the HP, UKHP, and LP groups. Neurological deficit scores showed no significant difference at R2h among the groups (p=0.443). However, neurological deficit scores showed significant differences at R24h among the groups, the neurological deficits scores of rats in the LP group are significantly higher than that in the other groups. Compared with that of 2 hours after reperfusion, neurological deficit scores deteriorated in the LP group (p=0.047) but was improved in the NP, HP and UKHP groups (p=0.076, 0.002, 0.017, respectively) at 24 hours after reperfusion. The infarct volume in the HP group was apparently smaller than that in the LP group (p=0.006). There was indeed a tendency that HT occurred more frequently in the UKHP group (42.8%) than in the HP (25%) and LP (28.5%) groups. MMP-9 expression showed significant increase around the ischemic lesion areas of the UKHP group and significant decrease in the cortical areas of the LP and HP groups but no significant difference in the basal ganglia of rats of all groups. Mild elevation of blood pressure during reperfusion is supposed to improve neurological outcomes in rats following ischemia/reperfusion. The incidence of HT tended to increase with the elevation of blood pressure and the administration of urokinase. Immunohistochemitry analysis indicated that incidence of HT may correlate with excessive expression of MMP-9.

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

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.

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.

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.

Cell mediated therapeutics for cancer treatment: Tumor homing cells as therapeutic delivery vehicles

NASA Astrophysics Data System (ADS)

Balivada, Sivasai

Many cell types were known to have migratory properties towards tumors and different research groups have shown reliable results regarding cells as delivery vehicles of therapeutics for targeted cancer treatment. Present report discusses proof of concept for 1. Cell mediated delivery of Magnetic nanoparticles (MNPs) and targeted Magnetic hyperthermia (MHT) as a cancer treatment by using in vivo mouse cancer models, 2. Cells surface engineering with chimeric proteins for targeted cancer treatment by using in vitro models. 1. Tumor homing cells can carry MNPs specifically to the tumor site and tumor burden will decrease after alternating magnetic field (AMF) exposure. To test this hypothesis, first we loaded Fe/Fe3O4 bi-magnetic NPs into neural progenitor cells (NPCs), which were previously shown to migrate towards melanoma tumors. We observed that NPCs loaded with MNPs travel to subcutaneous melanoma tumors. After alternating magnetic field (AMF) exposure, the targeted delivery of MNPs by the NPCs resulted in a mild decrease in tumor size (Chapter-2). Monocytes/macrophages (Mo/Ma) are known to infiltrate tumor sites, and also have phagocytic activity which can increase their uptake of MNPs. To test Mo/Ma-mediated MHT we transplanted Mo/Ma loaded with MNPs into a mouse model of pancreatic peritoneal carcinomatosis. We observed that MNP-loaded Mo/Ma infiltrated pancreatic tumors and, after AMF treatment, significantly prolonged the lives of mice bearing disseminated intraperitoneal pancreatic tumors (Chapter-3). 2. Targeted cancer treatment could be achieved by engineering tumor homing cell surfaces with tumor proteases cleavable, cancer cell specific recombinant therapeutic proteins. To test this, Urokinase and Calpain (tumor specific proteases) cleavable; prostate cancer cell (CaP) specific (CaP1 targeting peptide); apoptosis inducible (Caspase3 V266ED3)- rCasp3V266ED3 chimeric protein was designed in silico. Hypothesized membrane anchored chimeric protein (rCasp3V266ED3, rMcherry red) plasmids were constructed. Membrane anchoring and activity of designed proteins were analyzed in RAW264.7 Mo/Ma and HEK293 cells in vitro. Further, Urokinase (uPA) mediated cleavage and release of rCasp3V266ED3 from engineered cells was tested (Chapter-4). Animal models for cancer therapy are invaluable for preclinical testing of potential cancer treatments. Final chapter of present report shows evidence for immune-deficient line of pigs as a model for human cancers (Chapter-5)

An improved electronic twist-drill craniostomy procedure with post-operative urokinase instillation in treating chronic subdural hematoma.

PubMed

Lu, Jing; Shen, Dongwei; Hu, Fangjin; Zhou, Jianjun; Lan, Folin; Guo, Dongbing; Liu, Tianqing

2015-09-01

Twist-drill craniostomy (TDC) with closed-system drainage is a less invasive surgical technique for the treatment of chronic subdural hematoma (CSDH), but results in a higher disease recurrence rate. Therefore, we aimed to modify the TDC procedure in order to reduce the recurrence rate and further decrease complications. We retrospectively reviewed 230 cases of standard CSDH in 202 patients treated in our hospital between January 2006 and December 2013. We employed a new TDC device called micro-steel-needle-tube-bit for puncture and drainage. We chose an entry point 0.5cm anterior to the coronal suture at the superior temporal line and maintained post-operative drainage with urokinase instillation into the hematoma cavity. Clinical performance was assessed and compared by the Markwalder Grading Scale (MGS) score during the pre-operative period and at discharge. Recurrence of CSDH and complications were also recorded. Mean operation time was only 8.9 (6-13) min. Mean catheter indwelling duration and hospital stay were 1.18 (271/230, 1-3) and 2.27 (458/202, 2-9) days, respectively. The average frequency of urokinase instillation was 1.14 (262/230, 1-3) times. Intracerebral and sub-arachnoid hemorrhages were found in one patient, but were not in the puncture pathway. No perioperative deaths occurred. Recurrence was observed in only one patient at 28 days post-operation. Among 202 patients, 193 (95.54%) showed improved clinical symptoms and neurological function, and significantly lower MGS scores at discharge than pre-operation (0.13±0.45 vs. 1.37±0.55, P<0.01). In conclusion, electric TDC with micro-steel-needle-tube-bit at the pre-coronal suture entry point might be a safer, simpler, and faster mini-invasive surgical procedure for CSDH treatment. Post-operative drainage with instillation of urokinase could dramatically shorten drainage time and decrease recurrence rate. Copyright © 2015 Elsevier B.V. All rights reserved.

Kidney cell electrophoresis

NASA Technical Reports Server (NTRS)

Todd, P. W.

1985-01-01

Tasks were undertaken in support of two objectives. They are: (1) to carry out electrophoresis experiments on cells in microgravity; and (2) assess the feasibility of using purified kidney cells from embryonic kidney cultures as a source of important cell products. Investigations were carried out in the following areas: (1) ground based electrophoresis technology; (2) cell culture technology; (3) electrophoresis of cells; (4) urokinase assay research; (5) zero-g electrophoresis; and (6) flow cytometry.

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.

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

Electrophoretic mobilities of cultured human embryonic kidney cells in various buffers

NASA Technical Reports Server (NTRS)

1985-01-01

Data on the electrophoretic mobility distributions of cells in the new D-1 buffer and the interlaboratory standardization of urokinase assay methods are presented. A table of cell strains and recent data on cell dispersal methods are also included. It was decided that glycerol in A-1 electrophoretic mobility data on cultured human embryonic kidney cells subjected to electrophoresis in this buffer. The buffer composition is presented.

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.

Urokinase Receptor Counteracts Vascular Smooth Muscle Cell Functional Changes Induced by Surface Topography

PubMed Central

Kiyan, Yulia; Kurselis, Kestutis; Kiyan, Roman; Haller, Hermann; Chichkov, Boris N.; Dumler, Inna

2013-01-01

Current treatments for human coronary artery disease necessitate the development of the next generations of vascular bioimplants. Recent reports provide evidence that controlling cell orientation and morphology through topographical patterning might be beneficial for bioimplants and tissue engineering scaffolds. However, a concise understanding of cellular events underlying cell-biomaterial interaction remains missing. In this study, applying methods of laser material processing, we aimed to obtain useful markers to guide in the choice of better vascular biomaterials. Our data show that topographically treated human primary vascular smooth muscle cells (VSMC) have a distinct differentiation profile. In particular, cultivation of VSMC on the microgrooved biocompatible polymer E-shell induces VSMC modulation from synthetic to contractile phenotype and directs formation and maintaining of cell-cell communication and adhesion structures. We show that the urokinase receptor (uPAR) interferes with VSMC behavior on microstructured surfaces and serves as a critical regulator of VSMC functional fate. Our findings suggest that microtopography of the E-shell polymer could be important in determining VSMC phenotype and cytoskeleton organization. They further suggest uPAR as a useful target in the development of predictive models for clinical VSMC phenotyping on functional advanced biomaterials. PMID:23843899

Leukocyte- and endothelial-derived microparticles: a circulating source for fibrinolysis

PubMed Central

Lacroix, Romaric; Plawinski, Laurent; Robert, Stéphane; Doeuvre, Loïc; Sabatier, Florence; Martinez de Lizarrondo, Sara; Mezzapesa, Anna; Anfosso, Francine; Leroyer, Aurelie S.; Poullin, Pascale; Jourde, Noémie; Njock, Makon-Sébastien; Boulanger, Chantal M.; Anglés-Cano, Eduardo; Dignat-George, Françoise

2012-01-01

Background We recently assigned a new fibrinolytic function to cell-derived microparticles in vitro. In this study we explored the relevance of this novel property of microparticles to the in vivo situation. Design and Methods Circulating microparticles were isolated from the plasma of patients with thrombotic thrombocytopenic purpura or cardiovascular disease and from healthy subjects. Microparticles were also obtained from purified human blood cell subpopulations. The plasminogen activators on microparticles were identified by flow cytometry and enzyme-linked immunosorbent assays; their capacity to generate plasmin was quantified with a chromogenic assay and their fibrinolytic activity was determined by zymography. Results Circulating microparticles isolated from patients generate a range of plasmin activity at their surface. This property was related to a variable content of urokinase-type plasminogen activator and/or tissue plasminogen activator. Using distinct microparticle subpopulations, we demonstrated that plasmin is generated on endothelial and leukocyte microparticles, but not on microparticles of platelet or erythrocyte origin. Leukocyte-derived microparticles bear urokinase-type plasminogen activator and its receptor whereas endothelial microparticles carry tissue plasminogen activator and tissue plasminogen activator/inhibitor complexes. Conclusions Endothelial and leukocyte microparticles, bearing respectively tissue plasminogen activator or urokinase-type plasminogen activator, support a part of the fibrinolytic activity in the circulation which is modulated in pathological settings. Awareness of this blood-borne fibrinolytic activity conveyed by microparticles provides a more comprehensive view of the role of microparticles in the hemostatic equilibrium. PMID:22733025

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

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.

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.

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.

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.

Kidney Cell Electrophoresis

NASA Technical Reports Server (NTRS)

Todd, P.

1985-01-01

Materials and procedures for microgravity electrophoresis of living human embryonic kidney cells were evaluated, ground support in the form of analytical cell electrophoresis and flow cytometry was provided and cells returned from space flight were analyzed. Preflight culture media, electrophoresis buffer, fraction collection media, temperature profiles, and urokinase assay procedures were tested prior to flight. Electrophoretic mobility distributions of aliquots of the cell population to be fractionated in flight were obtained. The protocol established and utilized is given.

Epidermal growth factor- and hepatocyte growth factor-receptor activity in serum-free cultures of human hepatocytes.

PubMed

Runge, D M; Runge, D; Dorko, K; Pisarov, L A; Leckel, K; Kostrubsky, V E; Thomas, D; Strom, S C; Michalopoulos, G K

1999-02-01

Serum-free primary cultures of hepatocytes are a useful tool to study factors triggering hepatocyte proliferation and regeneration. We have developed a chemically defined serum-free system that allows human hepatocyte proliferation in the presence of epidermal growth factor and hepatocyte growth factor. DNA synthesis and accumulation were determined by [3H]thymidine incorporation and fluorometry, respectively. Western blot analyses and co-immunoprecipitations were used to investigate the association of proteins involved in epidermal growth factor and hepatocyte growth factor activation and signaling: epidermal growth factor receptor, hepatocyte growth factor receptor (MET), urokinase-type plasminogen activator and its receptor, and a member of the signal transducer and activator of transcription family, STAT-3. Primary human hepatocytes proliferated under serum-free conditions in a chemically defined medium for up to 12 days. Epidermal growth factor-receptor and MET were present and functional, decreasing over time. MET, urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor co-precipitated to varying degrees during the culture period. STAT-3 co-precipitated with epidermal growth factor-receptor and MET to varying degrees. Proliferation of human hepatocytes can improve by modification of a chemically defined medium originally used for rat hepatocyte cultures. In these long-term cultures of human hepatocytes, hepatocyte growth factor and epidermal growth factor can stimulate growth and differentiation by interacting with their receptors and initiating downstream signaling. This involves complex formation of the receptors with other plasma membrane components for MET (urokinase-type plasminogen activator in context of its receptor) and activation of STAT-3 for both receptors.

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.

Summary electrophoretic data base on human embryonic kidney cell strain 8514

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Arquiza, M. V.; Morrison, D. R.; Todd, P. W.

1985-01-01

To properly plan the electrophoresis equipment verification test (EEVT) and continuous flow electrophoresis system (CFES) experiments with human embryonic kidney cells, first a candidate cell lot had to be chosen on the basis of electrophoretic heterogeneity, growth potential, cytogenetics, and urokinase production. Cell lot 8514 from MA Bioproducts, Inc. was chosen for this purpose, and several essential analytical electrophoresis experiments were performed to test its final suitability for these experiments.

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.

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

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.

A label-free photoelectrochemical biosensor for urokinase-type plasminogen activator detection based on a g-C3N4/CdS nanocomposite.

PubMed

Liu, Xing-Pei; Chen, Jing-Shuai; Mao, Chang-Jie; Niu, He-Lin; Song, Ji-Ming; Jin, Bao-Kang

2018-09-26

Herein, we established a novel ultrasensitive photoelectrochemical biosensor for detecting urokinase-type plasminogen activator (u-PA), based on a g-C 3 N 4 /CdS nanocomposite. The prepared nanocomposite was characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, ultraviolet-visible absorption spectroscopy, and Fourier transform infrared spectroscopy, thus indicating that the nanocomposite was prepared successfully. In the typical process, the prepared nanocomposite was deposited on the surface of a bare FTO electrode. After being air-dried, the g-C 3 N 4 /CdS nanocomposite modified electrode was successively incubated with antibody against urokinase-type plasminogen activator and the blocking agent BSA to produce a photoelectrochemical biosensor for u-PA. In the presence of target u-PA antigen, the photocurrent response of the prepared biosensor electrode decreased significantly. The proposed novel photoelectrochemical biosensor exhibited good sensitivity, specificity, and reproducibility for u-PA detection, and a low detection limit of 33 fg mL -1 , ranging from 1 μg mL -1 -0.1 pg mL -1 . The proposed strategy should provide a promising method for detection of other biomarkers. Copyright © 2018 Elsevier B.V. All rights reserved.

Urokinase-receptor (u-PAR): an essential player in multiple games of cancer: a review on its role in tumor progression, invasion, metastasis, proliferation/dormancy, clinical outcome and minimal residual disease.

PubMed

Laufs, Stephanie; Schumacher, Jens; Allgayer, Heike

2006-08-01

The relevance of the u-PA system in mediating tumor-associated proteolysis, invasion and metastasis, amongst other phenomena associated with tumor progression, has been clearly demonstrated in diverse cancer entities. This review will update on the biological and clinical relevance of the urokinase-receptor (u-PAR). Specifically, the article focuses on the potential importance of u-PAR for the development of minimal residual disease in solid cancer, and in this context reviews the biological relevance of the u-PAR for tumor cell dormancy. Furthermore, transcriptional mechanisms regulating u-PAR in vitro and in vivo, and their potential clinical and therapeutic relevance in gastrointestinal cancers, are elucidated.

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.

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.

Characteristics of the level-of-evidence-1 disease forecast cancer biomarkers uPA and its inhibitor PAI-1.

PubMed

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

2010-10-01

In cancer, the serine protease urokinase-type plasminogen activator, its inhibitor (plasminogen activator inhibitor type-1) and the receptor (CD87), among other proteolytic factors, are involved in tumor cell dissemination and turnover of the extracellular matrix. Unsurprisingly, a battery of very uniform data, amassed since the end of the 1990s, has put these members of the plasminogen activation system into the forefront of prognostic/predictive cancer biomarkers relevant to predict the clinical course of cancer patients and their response to cancer therapy. The present review focuses on the molecular characteristics of the disease forecast biomarkers urokinase-type plasminogen activator and plasminogen activator inhibitor type-1, and techniques to quantitatively assess these cancer biomarkers, in the context of potential clinical application and personalized disease management.

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.

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

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.

The calcineurin–NFAT pathway allows for urokinase receptor-mediated beta3 integrin signaling to cause podocyte injury

PubMed Central

Shi, Wei; Ma, Juan; Sloan, Alexis; Faul, Christian; Wei, Changli; Reiser, Jochen; Yang, Yun; Liu, Shuangxin; Wang, Wenjian

2014-01-01

Circulating and podocyte-bound urokinase receptor (uPAR) is a mediator of podocyte injury, proteinuria, and focal segmental glomerulosclerosis (FSGS) allowing pathological activation of the uPAR-β3 integrin signaling axis. Clinically, calcineurin inhibitors (e.g., cyclosporine A, CsA) are known to suppress T cells, yet are also being used to reduce proteinuria in FSGS, suggesting the possibility of signal cross talk between uPAR and calcineurin. Calcineurin is known to facilitate the nuclear translocation of the nuclear factor of activated T cells (NFAT). Accordingly, in vivo conditional NFATc1 activation in podocytes leads to proteinuria in mice, yet the downstream targets of NFAT remain unclear. Here, we show that inducible podocyte-specific expression of constitutively active NFATc1 increased podocyte uPAR expression by binding to the Plaur gene promoter (encoding uPAR) in chromatin immunoprecipitation assays. Pathological uPAR signals in podocytes are independent of T cells and affect cell motility via activation, but not expression, changes of the β3 integrin and can be blocked by CsA, NFAT-siRNA, or the cell-permeable NFAT inhibitor (11R-VIVIT) using rodent models of glomerular disease (LPS; 5/6 nephrectomized rats). Taken together, these findings identify podocyte uPAR as a downstream target of NFAT and provide further insights into the pathogenesis of FSGS. PMID:23015147

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

Hemangiosarcoma and its cancer stem cell subpopulation are effectively killed by a toxin targeted through epidermal growth factor and urokinase receptors.

PubMed

Schappa, Jill T; Frantz, Aric M; Gorden, Brandi H; Dickerson, Erin B; Vallera, Daniel A; Modiano, Jaime F

2013-10-15

Targeted toxins have the potential to overcome intrinsic or acquired resistance of cancer cells to conventional cytotoxic agents. Here, we hypothesized that EGFuPA-toxin, a bispecific ligand-targeted toxin (BLT) consisting of a deimmunized Pseudomonas exotoxin (PE) conjugated to epidermal growth factor and urokinase, would efficiently target and kill cells derived from canine hemangiosarcoma (HSA), a highly chemotherapy resistant tumor, as well as cultured hemangiospheres, used as a surrogate for cancer stem cells (CSC). EGFuPA-toxin showed cytotoxicity in four HSA cell lines (Emma, Frog, DD-1 and SB) at a concentration of ≤100 nM, and the cytotoxicity was dependent on specific ligand-receptor interactions. Monospecific targeted toxins also killed these chemoresistant cells; in this case, a "threshold" level of EGFR expression appeared to be required to make cells sensitive to the monospecific EGF-toxin, but not to the monospecific uPA-toxin. The IC₅₀ of CSCs was higher by approximately two orders of magnitude as compared to non-CSCs, but these cells were still sensitive to EGFuPA-toxin at nanomolar (i.e., pharmacologically relevant) concentrations, and when targeted by EGFuPA-toxin, resulted in death of the entire cell population. Taken together, our results support the use of these toxins to treat chemoresistant tumors such as sarcomas, including those that conform to the CSC model. Our results also support the use of companion animals with cancer for further translational development of these cytotoxic molecules. Copyright © 2013 UICC.

Endotoxin-induced intravascular coagulation in rabbits: effect of tissue plasminogen activator vs urokinase of PAI generation, fibrin deposits and mortality.

PubMed

Paloma, M J; Páramo, J A; Rocha, E

1995-12-01

We have evaluated the effect of plasminogen activators (t-PA and urokinase) on an experimental model of disseminated intravascular coagulation (DIC) in rabbits by injection of 20 micrograms/kg/h of E. coli lipopolysaccharide during 6 h t-PA (0.2 mg/kg and 0.7 mg/kg), urokinase (3000 U/kg/h) and saline (control) were given simultaneously with endotoxin. Results indicated that urokinase and low dose of t-PA significantly reduced the increase of plasminogen activator inhibitor (PAI) activity observed 2 h after endotoxin (p < 0.001). High t-PA dose also diminished the PAI levels at 6 h (p < 0.001). A significant reduction of fibrin deposits in kidneys was observed din both t-PA treated groups as compared with findings in the group of rabbits infused with saline solution (p < 0.005), whereas urokinase had no significant effect on the extent of fibrin deposition. Finally, the mortality rate in the control group (70%) was reduced to 50% in rabbits receiving high doses of t-PA. In conclusion, treatment with t-PA resulted in reduced PAI generation, fibrin deposits and mortality in endotoxin-treated rabbits.

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

Use of intrathecal urokinase in repeated shunt and external ventricular drain blockage from high CSF protein due to an optic pathway glioma.

PubMed

Shooman, David; Vajramani, Girish V; Davidson, Jennifer; Sparrow, Owen C

2010-05-01

High cerebrospinal fluid (CSF) protein is a recognised association of optic pathway gliomas. This can occlude ventriculoperitoneal (VP) shunt catheters or external ventricular drains (EVD). We describe an 8-year-old boy with an optic pathway glioma, who had frequent episodes of a blocked VP shunt and EVD due to high CSF protein level. This responded favourably to repeated urokinase instillation into the catheter lumen. We present the course of treatment and the method of administration and review the literature behind thrombolytic therapy for occluded catheter devices. Intrathecal urokinase was effective in unblocking the EVD and lysing the protein clots within the ventricle. We found no previously published cases of intrathecal urokinase for this problem. This novel intervention seemed promising as a safe and effective means of maintaining EVD patency in cases complicated by excessive protein loads in CSF and hydrocephalus.

Quebec platelet disorder: features, pathogenesis and treatment.

PubMed

Diamandis, Maria; Veljkovic, D Kika; Maurer-Spurej, Elisabeth; Rivard, Georges E; Hayward, Catherine P M

2008-03-01

Quebec platelet disorder (QPD) is a rare, autosomal-dominant, inherited bleeding disorder that is associated with unique abnormalities in fibrinolysis. Its hallmark features are delayed-onset bleeding following hemostatic challenges that responds to fibrinolytic inhibitor therapy and increased expression and storage of the fibrinolytic enzyme urokinase plasminogen activator in platelets, without increased plasma urokinase plasminogen activator or systemic fibrinolysis. The increased urokinase plasminogen activator in QPD platelets is only partially inhibited, and, as a result, there is intraplatelet generation of plasmin, and secondary degradation of many platelet alpha-granule proteins. During clot formation, the urokinase plasminogen activator released by QPD platelets leads to platelet-dependent increased fibrinolysis, and this is postulated to be a major contributor to QPD bleeding. The focus of the present review is to summarize the current state of knowledge on QPD, including the history of this disorder, its clinical and laboratory features, and recommended approaches for its diagnosis and treatment.

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.

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

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.

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.

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.

[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.

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

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

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.

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

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).

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.

Superselective Urokinase Infusion Therapy for Dorsalis Pedis Artery Occlusion in Buerger's Disease

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

Kubota, Yasushi; Kichikawa, Kimihiko; Uchida, Hideo

1997-09-15

Occlusion of the proximal left dorsalis pedis artery (DPA) in a patient with Buerger's disease was treated by continuous urokinase intraarterial infusion using a microcatheter. Recanalization of the DPA and healing of a toe ulcer were achieved. The patient remains asymptomatic during a 4-year follow-up.

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.

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

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.

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

A New Class of Orthosteric uPAR•uPA Small-Molecule Antagonists Are Allosteric Inhibitors of the uPAR•Vitronectin Interaction

PubMed Central

Liu, Degang; Zhou, Donghui; Wang, Bo; Knabe, William Eric; Meroueh, Samy O.

2015-01-01

The urokinase receptor (uPAR) is a GPI-anchored cell surface receptor that is at the center of an intricate network of protein-protein interactions. Its immediate binding partners are the serine proteinase urokinase (uPA), and vitronectin (VTN), a component of the extracellular matrix. uPA and VTN bind at distinct sites on uPAR to promote extracellular matrix degradation and integrin signaling, respectively. Here, we report the discovery of a new class of pyrrolone small-molecule inhibitors of the tight ∼1 nM uPAR•uPA protein-protein interaction. These compounds were designed to bind to the uPA pocket on uPAR. The highest affinity compound, namely 7, displaced a fluorescently-labeled α-helical peptide (AE147-FAM) with an inhibition constant Ki of 0.7 µM and inhibited the tight uPAR•uPAATF interaction with an IC50 of 18 µM. Biophysical studies with surface plasmon resonance showed that VTN binding is highly dependent on uPA. This cooperative binding was confirmed as 7, which binds at the uPAR•uPA interface, also inhibited the distal VTN•uPAR interaction. In cell culture, 7 blocked the uPAR•uPA interaction in uPAR-expressing human embryonic kidney (HEK-293) cells, and impaired cell adhesion to VTN, a process that is mediated by integrins. As a result, 7 inhibited integrin signaling in MDA-MB-231 cancer cells as evidenced by a decrease in focal adhesion kinase (FAK) phosphorylation and Rac1 GTPase activation. Consistent with these results, 7 blocked breast MDA-MB-231 cancer cell invasion with IC50 values similar to those observed in ELISA and surface plasmon resonance competition studies. Explicit-solvent molecular dynamics simulations show that the cooperativity between uPA and VTN is attributed to stabilization of uPAR motion by uPA. In addition, free energy calculations revealed that uPA stabilizes the VTN•uPARSMB interaction through more favorable electrostatics and entropy. Disruption of the uPAR•VTNSMB interaction by 7 is consistent with the cooperative binding to uPAR by uPA and VTN. Interestingly, the VTNSMB•uPAR interaction was less favorable in the VTNSMB•uPAR•7 complex suggesting potential cooperativity between 7 and VTN. Compound 7 provides an excellent starting point for the development of more potent derivatives to explore uPAR biology. PMID:25671694

A new class of orthosteric uPAR·uPA small-molecule antagonists are allosteric inhibitors of the uPAR·vitronectin interaction.

PubMed

Liu, Degang; Zhou, Donghui; Wang, Bo; Knabe, William Eric; Meroueh, Samy O

2015-06-19

The urokinase receptor (uPAR) is a GPI-anchored cell surface receptor that is at the center of an intricate network of protein-protein interactions. Its immediate binding partners are the serine proteinase urokinase (uPA), and vitronectin (VTN), a component of the extracellular matrix. uPA and VTN bind at distinct sites on uPAR to promote extracellular matrix degradation and integrin signaling, respectively. Here, we report the discovery of a new class of pyrrolone small-molecule inhibitors of the tight ∼1 nM uPAR·uPA protein-protein interaction. These compounds were designed to bind to the uPA pocket on uPAR. The highest affinity compound, namely 7, displaced a fluorescently labeled α-helical peptide (AE147-FAM) with an inhibition constant Ki of 0.7 μM and inhibited the tight uPAR·uPAATF interaction with an IC50 of 18 μM. Biophysical studies with surface plasmon resonance showed that VTN binding is highly dependent on uPA. This cooperative binding was confirmed as 7, which binds at the uPAR·uPA interface, also inhibited the distal VTN·uPAR interaction. In cell culture, 7 blocked the uPAR·uPA interaction in uPAR-expressing human embryonic kidney (HEK-293) cells and impaired cell adhesion to VTN, a process that is mediated by integrins. As a result, 7 inhibited integrin signaling in MDA-MB-231 cancer cells as evidenced by a decrease in focal adhesion kinase (FAK) phosphorylation and Rac1 GTPase activation. Consistent with these results, 7 blocked breast MDA-MB-231 cancer cell invasion with IC50 values similar to those observed in ELISA and surface plasmon resonance competition studies. Explicit-solvent molecular dynamics simulations show that the cooperativity between uPA and VTN is attributed to stabilization of uPAR motion by uPA. In addition, free energy calculations revealed that uPA stabilizes the VTNSMB·uPAR interaction through more favorable electrostatics and entropy. Disruption of the uPAR·VTNSMB interaction by 7 is consistent with the cooperative binding to uPAR by uPA and VTN. Interestingly, the VTNSMB·uPAR interaction was less favorable in the VTNSMB·uPAR·7 complex suggesting potential cooperativity between 7 and VTN. Compound 7 provides an excellent starting point for the development of more potent derivatives to explore uPAR biology.

KSC-03pd1458

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - A.K. Love, with Instrumentation Technology Associates, Inc., displays one of the boxes used for cancer cell research, an experiment carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

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

KSC-03PD-1458

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - A.K. Love, with Instrumentation Technology Associates, Inc., displays one of the boxes used for cancer cell research, an experiment carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

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

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

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.

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.

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.

High dose urokinase for restoration of patency of occluded permanent central venous catheters in hemodialysis patients.

PubMed

Shavit, L; Lifschitz, M; Plaksin, J; Grenader, T; Slotki, I

2010-10-01

Catheter thrombosis is common and results in inadequate dialysis treatment and, frequently, in catheter loss. Since dialysis treatment runs on a strict schedule, occluded catheters need to be restored in a timely and cost effective manner. We present a new shortened protocol of urokinase infusion that allows hemodialysis to be performed within 90 minutes. To chronic hemodialysis patients, who developed complete catheter occlusion, urokinase was infused simultaneously through both lumens of the catheter (125,000 units to each lumen) over 90 minutes. Technical success was defined as restoring blood pump speed to at least 250 ml/min. We determined the average time from catheter placement to first clot event (primary patency PP), recurrent clot event after urokinase treatment (secondary patency SP), catheter salvage rate and cause for removal. 37 catheters developed total thrombosis and urokinase was used to restore patency one or more times (total 47 treatments). Catheter salvage rate was 97 %. The average time of PP was 152 ± 56 days (7 - 784 days). Nine patients (30%) developed recurrent occlusion and the average time of SP was 64 ± 34 days (2 - 364 days). One catheter was removed because of dysfunction due to thrombosis. Other catheters were removed due to infection, fistula maturation or fell out spontaneously. Hemodialysis was performed immediately after treatment with blood speed of 250 ml/min in all patients. Our protocol is highly effective, short, and allows to restore patency of totally occluded central venous catheters with minimal disruption of the dialysis session.

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.

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

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 tight junction protein ZO-2 and Janus kinase 1 mediate intercellular communications in vascular smooth muscle cells

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

Tkachuk, Natalia; Tkachuk, Sergey; Patecki, Margret

2011-07-08

Highlights: {yields} The tight junction protein ZO-2 associates with Jak1 in vascular smooth muscle cells via ZO-2 N-terminal fragment. {yields} Jak1 mediates ZO-2 tyrosine phosphorylation and ZO-2 localization to the sites of homotypic intercellular contacts. {yields} The urokinase receptor uPAR regulates ZO-2/Jak1 functional association. {yields} The ZO-2/Jak1/uPAR signaling complex is required for vascular smooth muscle cells functional network formation. -- Abstract: Recent evidence points to a multifunctional role of ZO-2, the tight junction protein of the MAGUK (membrane-associated guanylate kinase-like) family. Though ZO-2 has been found in cell types lacking tight junction structures, such as vascular smooth muscle cells (VSMC),more » little is known about ZO-2 function in these cells. We provide evidence that ZO-2 mediates specific homotypic cell-to-cell contacts between VSMC. Using mass spectrometry we found that ZO-2 is associated with the non-receptor tyrosine kinase Jak1. By generating specific ZO-2 constructs we further found that the N-terminal fragment of ZO-2 molecule is responsible for this interaction. Adenovirus-based expression of Jak1 inactive mutant demonstrated that Jak1 mediates ZO-2 tyrosine phosphorylation. By means of RNA silencing, expression of Jak1 mutant form and fluorescently labeled ZO-2 fusion protein we further specified that active Jak1, but not Jak1 inactive mutant, mediates ZO-2 localization to the sites of intercellular contacts. We identified the urokinase receptor uPAR as a pre-requisite for these cellular events. Functional requirement of the revealed signaling complex for VSMC network formation was confirmed in experiments using Matrigel and in contraction assay. Our findings imply involvement of the ZO-2 tight junction independent signaling complex containing Jak1 and uPAR in VSMC intercellular communications. This mechanism may contribute to vascular remodeling in occlusive cardiovascular diseases and in arteriogenesis.« less

Intravenous Hyaluronidase with Urokinase as Treatment for Rabbit Retinal Artery Hyaluronic Acid Embolism.

PubMed

Chiang, ChengAn; Zhou, ShuangBai; Chen, ChuHsin; Ho, Dave S; Zhang, HuiZhong; Liu, Kai

2016-12-01

Although various salvage methods have been proposed to treat intraretinal artery hyaluronic acid embolism, their applications are still limited by various factors. The authors investigated the effectiveness of intravenous hyaluronidase with urokinase for resolving retinal artery hyaluronic acid embolism. The anatomy of rabbit ophthalmic and fundus arteries (retinal and choroid artery) was studied. Approximately 0.35 ml of hyaluronic acid was injected into the ophthalmic artery to create a retinal artery embolism model. The rabbits were grouped randomly (groups A, B, C, D, E, and F) and given hyaluronidase with urokinase intravenously at different postobstruction time points (10, 20, 30, 40, 50, and 60 minutes). Saline was given to the control group. Fundus vascular (retinal and choroid artery) reperfusion status and the effectiveness of the solution on the obstruction of each group were observed for 5 days. The animal model closely imitated actual hyaluronic acid ophthalmic/retinal artery obstructions. Three vascular conditions were observed after hyaluronidase with urokinase injection: total, partial, and no reperfusion. Groups A, B, and C showed a significantly higher overall solution effectiveness rate (total/partial reperfusion) compared with the control group (p = 0.001, p = 0.001, and p = 0.005, respectively). Solution effectiveness in groups D, E, and F showed no difference compared with the control group (p = 0.628, p = 1.000, and p = 1.000, respectively). The effectiveness of the solution drops dramatically if given after 30 minutes of obstruction. The authors' method can indeed help resolve retinal artery hyaluronic acid obstruction. Intravenous hyaluronidase with urokinase technique shows possible potential to become a standardized treatment protocol for intraretinal artery hyaluronic acid embolism with further clinical tests.

Reperfusion of occluded branch retinal arteries by transluminal Nd:YAG laser embolysis combined with intravenous thrombolysis of urokinase

PubMed Central

Chai, Fang; Du, Shanshuang; Zhao, Xiquan

2017-01-01

Purpose: To report successful treatment with transluminal Nd:YAG laser embolysis (TYE) combined with urokinase thrombolysis for reperfusion of occluded branch retinal arteries with visible emboli. Methods: A total of 34 eyes from 34 patients with acute, severe vision loss secondary to a branch retinal artery occlusion with visible emboli and retinal whitening were examined. Each patient was administered TYE therapy, which focused on the embolus, using an ocular contact lens; a 0.3–0.9 mJ laser pulse was delivered directly and gradually according to the reaction. Fundus photographs and fundus fluorescein angiography (FFA) were obtained before and immediately after the laser treatment. All patients received urokinase thrombolysis therapy drops intravenously for 5 days at 10–20 u/d. The follow-up period ranged from 6 to 14 months after therapy. The morphological characteristics of FFA associated with obstruction recovery of arterial fluorescence filling and visual function were analyzed. Results: After TYE therapy, FFA examinations showed that the retinal artery and its branches exhibited completely restored blood flow without obstruction in 13 eyes, accounting for 38.2% of the cases. The blood flow was mostly recovered in 11 eyes (32.4% of patients). FFA examinations following the combined intravenous urokinase thrombolysis therapy showed that the retinal artery and its branches exhibited completely restored blood flow after obstruction in 16 eyes (47.1% of patients). The blood flow was mostly recovered in 15 eyes (44.1% of patients). Conclusion: TYE combined with urokinase thrombolysis is effective for reperfusion of occluded branch retinal arteries and improving visual recovery in patients with visible emboli. PMID:29162667

Transradial Approach for Transcatheter Selective Superior Mesenteric Artery Urokinase Infusion Therapy in Patients with Acute Extensive Portal and Superior Mesenteric Vein Thrombosis

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

Wang Maoqiang, E-mail: wangmq@vip.sina.com; Guo Liping; Lin Hanying

2010-02-15

The purpose of this investigation was to assess the feasibility and effectiveness of transradial approach for transcatheter superior mesenteric artery (SMA) urokinase infusion therapy in patients with acute extensive portal and superior mesenteric venous thrombosis. During a period of 7 years, 16 patients with acute extensive thrombosis of the portal (PV) and superior mesenteric veins (SMV) were treated by transcatheter selective SMA urokinase infusion therapy by way of the radial artery. The mean age of the patients was 39.5 years. Through the radial sheath, a 5F Cobra catheter was inserted into the SMA, and continuous infusion of urokinase was performedmore » for 5-11 days (7.1 {+-} 2.5 days). Adequate anticoagulation was given during treatment, throughout hospitalization, and after discharge. Technical success was achieved in all 16 patients. Substantial clinical improvement was seen in these 16 patients after the procedure. Minor complications at the radial puncture site were observed in 5 patients, but trans-SMA infusion therapy was not interrupted. Follow-up computed tomography scan before discharge demonstrated nearly complete disappearance of PV-SMV thrombosis in 9 patients and partial recanalization of PV-SMV thrombosis in 7 patients. The 16 patients were discharged 9-19 days (12 {+-} 6.0 days) after admission. Mean duration of follow-up after hospital discharge was 44 {+-} 18.5 months, and no recurrent episodes of PV-SMV thrombosis developed during that time period. Transradial approach for transcatheter selective SMA urokinase infusion therapy in addition to anticoagulation is a safe and effective therapy for the management of patients with acute extensive PV-SMV thrombosis.« less

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.

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

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

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.

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.

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.

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.

The Fibrin slide assay for detecting urokinase activity in human fetal kidney cells

NASA Technical Reports Server (NTRS)

Sedor, K.

1985-01-01

The Fibrin Slide Technique of Hau C. Kwaan and Tage Astrup is discussed. This relatively simple assay involves two steps: the formation of an artificial clot and then the addition of an enzyme (UKOKINASE) to dissolve the clot. The actual dissolving away of the clot is detected by the appearance of holes (lysis zones) in the stained clot. The procedure of Kwaan and Astrup is repeated, along with modifications and suggestions for improvements based on experience with the technique.

Effects of lipoprotein(a) on thrombolysis.

PubMed

von Hodenberg, E; Pestel, E; Kreuzer, J; Freitag, M; Bode, C

1994-01-01

Lipoprotein(a) (Lp(a)) and plasminogen share a high degree of structural homology. Therefore it has been suggested that elevated levels of Lp(a) may inhibit the profibrinolytic activity at the cell surface and increase the risk of thrombosis by competitive inhibition of plasminogen. In the present study we evaluated whether high levels of Lp(a) affect thrombolytic therapy in patients with acute myocardial infarction. Forty-one patients with acute myocardial infarction were treated with a combination of recombinant tissue-type plasminogen activator and human single-chain urokinase-type plasminogen activator. Coronary patency was assessed angiographically 90 min after initiation of treatment. Thrombolysis was successful in 30 and unsuccessful in 11 patients. Patients with high Lp(a) levels (> 25 mg/dl) (n = 9) responded equally well to thrombolytic therapy (8 of 9, patency 89%) as did patients with normal or low levels of Lp(a) (22 of 32, patency 70%, difference P > 0.1). The results demonstrate that high levels of Lp(a) do not influence thrombolysis in patients with acute myocardial infarction when low-dose pharmacologic concentrations of recombinant tissue-type plasminogen activator and human single chain urokinase-type plasminogen activator are applied in combination.

Kidney cell electrophoresis, continuing task

NASA Technical Reports Server (NTRS)

Todd, P. W.

1985-01-01

Materials and procedures for microgravity electrophoresis of living human embryonic kidney cells were evaluated to provide ground support in the form of analytical cell electrophoresis and flow cytometry. Preflight culture media, electrophoresis buffer, fraction collection media, temperature profiles, and urokinase assay procedures were tested prior to flight. Electrophoretic mobility distributions of aliquots of the cell population to be fractionated in flight were obtained. Cells were prepared in suspension prior to flight in electrophoresis buffer and 10% calf serum. Electrophoretic separation proceeded in electrophoresis buffer without serum in the Continuous Flow Electrophoretic Separator, and fractions were collected into sample bags containing culture medium and concentrated serum. Fractions that yielded enough progeny cells were analyzed for morphology and electrophoretic mobility distributions. It is noted that the lowest mobility fraction studied produced higher mobility progeny while the other fractions produced progeny cells with mobilities related to the fractions from which they were collected.

Silk screen based dual spin-filter module for perfusion culture of adherent and non-adherent mammalian cells.

PubMed

Kamthan, Shweta; Gomes, James; Roychoudhury, Pradip K

2014-08-01

Spin-filters have been primarily used for producing therapeutic proteins from mammalian cells. However, disposability and/or high filter clogging of the existing spin-filter systems affect the process economy and productivity. Hence, to address these drawbacks a reusable dual spin-filter module for perfusion culture of adherent and non-adherent mammalian cells was designed. Two non-woven Bombyx mori silk layers were used as filter screen; the outer layer was conducive to cell attachment whilst the inner was non-conducive. Adherent cells can be cultured either in suspended mode using its inner single module or as monolayer of cells using its dual concentric module. We achieved 30 % higher urokinase productivity as compared to the stainless-steel spin-filter during perfusion experiments of adherent human kidney cells in suspended mode. This was due to the hydrophobic and negatively-charged silk screen that allows clog-free perfusion culture for prolonged periods.

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.

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.

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.

Extracellular Collagen Promotes Interleukin-1β-Induced Urokinase-Type Plasminogen Activator Production by Human Corneal Fibroblasts.

PubMed

Sugioka, Koji; Kodama-Takahashi, Aya; Yoshida, Koji; Aomatsu, Keiichi; Okada, Kiyotaka; Nishida, Teruo; Shimomura, Yoshikazu

2017-03-01

Keratocytes maintain homeostasis of the corneal stroma through synthesis, secretion, and degradation of collagen fibrils of the extracellular matrix. Given that these cells are essentially embedded in a collagen matrix, keratocyte-collagen interactions may play a key role in regulation of the expression or activation of enzymes responsible for matrix degradation including urokinase-type plasminogen activator (uPA), plasmin, and matrix metalloproteinases (MMPs). We examined the effect of extracellular collagen on the production of uPA by corneal fibroblasts (activated keratocytes) stimulated with the proinflammatory cytokine interleukin-1β (IL-1β). Human corneal fibroblasts were cultured either on plastic or in a three-dimensional gel of type I collagen. Plasminogen activators were detected by fibrin zymography, whereas the IL-1 receptor (IL-1R) and MMPs were detected by immunoblot analysis. Collagen degradation by corneal fibroblasts was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. Collagen and IL-1β synergistically increased the synthesis and secretion of uPA in corneal fibroblasts. Collagen also upregulated IL-1R expression in the cells in a concentration-dependent manner. The conversion of extracellular plasminogen to plasmin, as well as the plasminogen-dependent activation of MMP-1 and MMP-3 and degradation of collagen apparent in three-dimensional cultures of corneal fibroblasts exposed to IL-1β, were all abolished by a selective uPA inhibitor. Collagen and IL-1β cooperate to upregulate uPA production by corneal fibroblasts. Furthermore, IL-1β-induced collagen degradation by these cells appears to be strictly dependent on uPA expression and mediated by a uPA-plasmin-MMP pathway.

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

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.

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.

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.

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

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

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

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.

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.

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.

The Effectiveness of Subdural Drains Using Urokinase after Burr Hole Evacuation of Subacute Subdural Hematoma in Elderly Patients: A Prelimilary Report

PubMed Central

Yeo, Chang-Gi; Jeon, Woo-Yeol; Kim, Seong-Ho; Kim, Oh-Lyong

2016-01-01

Objective A subdural drain using urokinase after a burr hole hematoma evacuation was performed for subacute subdural hematoma (SASDH), and its effectiveness and safety in elderly patients were evaluated. Methods Between January 2013 and May 2015, subdural drains using urokinase after burr hole hematoma evacuation were performed in 19 elderly patients. The inclusion criteria were as follows: 1) a subdural hematoma occurring between 4 and 20 days after injury; 2) worsening neurological symptoms, from mild to moderate or severe, due to injury during the subacute stage; 3) a mix of solid clots (high-density lighter shadow) and fluid hematoma (low-density darker shadow) on the computed tomography (CT) scan; 4) a score of ≥9 on the Glasgow Coma Scale (GCS) assessed immediately before surgery; and 5) an age of ≥65 years. When the majority of the hematoma was evacuated on the CT, we removed the catheter. Results Under local anesthesia, a catheter was inserted into the hematoma through a burr hole. The mean age of the patients was 73.7 years (range, 65-87 years). The mean preoperative GCS score was 11.2 (range, 10-13), and the mean Glasgow Outcome Scale score for all patients was 5 at discharge. No recurrences of hematomas or surgical complications were observed. Conclusion A subdural drain using urokinase after burr hole hematoma evacuation under local anesthesia is thought to be an effective and safe method of blood clot removal with low morbidity. This surgical method is less invasive for treating elderly patients with SASDH. PMID:27857916

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

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

[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.

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

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., works on an experiment found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., works on an experiment found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto is one of the scientists recovering experiments found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto is one of the scientists recovering experiments found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

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.

Estradiol attenuates EGF-induced rapid uPAR mobilization and cell migration via the G-protein-coupled receptor 30 in ovarian cancer cells.

PubMed

Henic, Emir; Noskova, Vera; Høyer-Hansen, Gunilla; Hansson, Stefan; Casslén, Bertil

2009-02-01

Epidermal growth factor (EGF) stimulates proliferation and migration in ovarian cancer cells, and high tumor expression of the EGF system correlates with poor prognosis. Epidermal growth factor upregulates urokinase plasminogen activator receptor (uPAR) on the cell surface via 3 distinct mechanisms: rapid mobilization of uPAR from detergent-resistant domains, increased mRNA, and decreased degradation. G-protein-coupled receptor 30 (GPR30) is a newly identified membrane estrogen receptor (ER).The objective of this study was to explore the effects of 17beta-estradiol (E(2)) on uPAR expression and cell migration in ovarian cancer cells and further to identify the ER involved.We used 7 ovarian cancer cell lines, cell migration assay, cellular binding of (125)I-uPA, cellular degradation of (125)I-uPA/PAI-1 complex, enzyme-linked immunosorbent assay for uPAR, solid-phase enzyme immunoassay for ERalpha, and quantitative polymerase chain reaction. Estradiol attenuates the stimulatory effect of EGF on cell migration and uPAR expression. Specifically, E(2) reduces the very rapid increase of detergent extractable uPAR, which occurs within minutes of EGF stimulation and probably represents mobilization of uPAR from detergent-resistant domains such as lipid rafts. Estradiol influenced neither the amount of uPAR mRNA nor the rate of uPAR degradation or solubilization. The nuclear ER antagonists ICI 182780 and tamoxifen, which are GPR30 agonists, as well as the specifically constructed GPR30 agonist G1, mimicked the effect of E(2) on uPAR expression and cell migration. OVCAR-3 cells express mRNA for GPR30.Estradiol attenuates EGF-induced mobilization of ligated uPAR from detergent-resistant domains and subsequent migration in ovarian cancer cells. The response to various ER ligands indicates that this effect is mediated via the membrane ER GPR30.

A Less Invasive Strategy for Ruptured Cerebral Aneurysms with Intracerebral Hematomas: Endovascular Coil Embolization Followed by Stereotactic Aspiration of Hematomas Using Urokinase

PubMed Central

Kim, Sang Heum; Kong, Min Ho

2017-01-01

Objective Aneurysm clipping and simultaneous hematoma evacuation through open craniotomy is traditionally recommended for ruptured cerebral aneurysms accompanied by intracerebral or intrasylvian hemorrhages. We report our experience of adapting a less invasive treatment strategy in poor-grade patients with intracerebral or intrasylvian hemorrhages associated with ruptured cerebral aneurysms, where the associated ruptured cerebral aneurysms were managed by endovascular coil embolization, followed by stereotactic aspiration of hematomas (SRH) using urokinase. Materials and Methods We retrospectively analyzed 112 patients with ruptured cerebral aneurysms. There were accompanying intracerebral or intrasylvian hemorrhages in 36 patients (32.1%). The most common site for these ruptured aneurysms was the middle cerebral artery (MCA) (n = 15; 41.6%). Endovascular coil embolization followed by SRH using urokinase was performed in 9 patients (25%). Results In these 9 patients, the most common site of aneurysms was the MCA (n = 3; 33.4%); the hematoma volume ranged from 19.24 to 61.68 mL. Four patients who were World Federation of Neurological Surgeons (WFNS) grade-IV on admission, achieved favorable outcomes (Glasgow Outcome Score [GOS] 4 or 5) at 6-months postoperatively. In the five patients who were WFNS grade-V on admission, one achieved a favorable outcome, whereas 4 achieved GOS scores of 2 or 3, 6-months postoperatively. There was no mortality. Conclusion If immediate hematoma evacuation is not mandated by clinical or radiological signs of brain herniation, a less invasive strategy, such as endovascular coil embolization followed by SRH using urokinase, may be a good alternative in poor-grade patients with intracerebral or intrasylvian hemorrhages associated with ruptured cerebral aneurysms. PMID:29152466

Fibrin sheaths in central venous port catheters: treatment with low-dose, single injection of urokinase on an outpatient basis.

PubMed

Chang, De-Hua; Mammadov, Kamal; Hickethier, Tilman; Borggrefe, Jan; Hellmich, Martin; Maintz, David; Kabbasch, Christoph

2017-01-01

Evaluation of the efficacy of single-shot, low-dose urokinase administration for the treatment of port catheter-associated fibrin sheaths. Forty-six patients were retrospectively evaluated for 54 episodes of port catheter dysfunction. The presence of a fibrin sheath was detected by angiographic contrast examinations. On an outpatient basis, patients subsequently received thrombolysis consisting of a single injection of urokinase (15.000 IU in 1.5 mL normal saline) through the port system. A second attempt was made in cases of treatment failure. Patients were followed up for technical success, complications and long-term outcome. Port dysfunction occurred at a median of 117 days after implantation (range: 7-825 days). The technical success after first port dysfunction by thrombolysis was 87% (40/46); thereof, initial thrombolysis was effective in 78% (36/46). Nine patients (20%) received a second dose of urokinase after previous treatment failure. Follow-up was available for 26 of 40 patients after successful thrombolysis. In 8 of these, rethrombosis occurred after a median of 98 days (range: 21-354 days), whereby rethrombolysis was effective in 5 of 7 (63%) patients. The overall success of all thrombolyses performed was 70% (45/64). No procedure-related technical or clinical complications occurred. After first favorable thrombolysis, a Kaplan-Meier analysis yielded a 30-, 90- and 180-day probability of patency of 96%, 87% and 81%. Thrombolytic therapy on an outpatient basis appears to be a safe and efficient. Three-month patency rates are comparable to more invasive treatment options, including catheter exchange over a guide wire and percutaneous fibrin sheath stripping.

Fibrin sheaths in central venous port catheters: treatment with low-dose, single injection of urokinase on an outpatient basis

PubMed Central

Chang, De-Hua; Mammadov, Kamal; Hickethier, Tilman; Borggrefe, Jan; Hellmich, Martin; Maintz, David; Kabbasch, Christoph

2017-01-01

Purpose Evaluation of the efficacy of single-shot, low-dose urokinase administration for the treatment of port catheter-associated fibrin sheaths. Methods Forty-six patients were retrospectively evaluated for 54 episodes of port catheter dysfunction. The presence of a fibrin sheath was detected by angiographic contrast examinations. On an outpatient basis, patients subsequently received thrombolysis consisting of a single injection of urokinase (15.000 IU in 1.5 mL normal saline) through the port system. A second attempt was made in cases of treatment failure. Patients were followed up for technical success, complications and long-term outcome. Results Port dysfunction occurred at a median of 117 days after implantation (range: 7–825 days). The technical success after first port dysfunction by thrombolysis was 87% (40/46); thereof, initial thrombolysis was effective in 78% (36/46). Nine patients (20%) received a second dose of urokinase after previous treatment failure. Follow-up was available for 26 of 40 patients after successful thrombolysis. In 8 of these, rethrombosis occurred after a median of 98 days (range: 21–354 days), whereby rethrombolysis was effective in 5 of 7 (63%) patients. The overall success of all thrombolyses performed was 70% (45/64). No procedure-related technical or clinical complications occurred. After first favorable thrombolysis, a Kaplan–Meier analysis yielded a 30-, 90- and 180-day probability of patency of 96%, 87% and 81%. Conclusion Thrombolytic therapy on an outpatient basis appears to be a safe and efficient. Three-month patency rates are comparable to more invasive treatment options, including catheter exchange over a guide wire and percutaneous fibrin sheath stripping. PMID:28182117

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.

[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.

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.

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.

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.

Drilling skull plus injection of urokinase in the treatment of epidural haematoma: a preliminary study.

PubMed

Liu, Weiguo; Ma, Li; Wen, Liang; Shen, Fang; Sheng, Hansong; Zhou, Bo; Hu, Weiwei; Zheng, Xiujue; Yang, Xiaofeng

2008-02-01

This study was performed to evaluate the effectiveness of a minimally invasive approach to manage patients with epidural haematoma (EDH). The surgical indication and key points were investigated. Descriptive, retrospective study. Twenty-one patients with traumatic EDH were treated through the following method: After anaesthesia, twist drill trepanations were performed followed by a placement of drainage tubes. Twenty ku urokinase in 3 ml saline was injected into the haematoma cyst through the tube, which was closed for 3 hours before connection to a vacuum ball with negative pressure. The injection was repeated three times a day after operation. The fibrinolytic agents were not used in the 'acute group' because of the risk of rebleeding. CT scans were performed according to the changes of clinical manifestations. The drainage tubes were left for 3-5 days before most clots were resolved. The patients discharged after 7 days' hospitalization on average. No infections or recurrence of EDHs were observed in this series. Drilling skull plus injection of urokinase through drainage tube is a safe and effective method with less injury in the treatment of a selected part of patients with EDHs.

A Biological Tissue Adhesive and Dissolvent System for Intraocular Tumor Plaque Brachytherapy.

PubMed

Zloto, Ofira; Vishnevskia-Dai, Vicktoria; Moisseiev, Joseph; Belkin, Michael; Fabian, Ido Didi

2016-02-01

To examine a novel technique for simplified placement and removal of plaque brachytherapy by fibrin glue and urokinase (medac Gmbh, Hamburg, Germany). In six enucleated porcine eyes, plaques were placed on the episclera and fibrin glue was applied to cover it. Urokinase was used to dissolve the glue in three eyes and saline was used in three eyes. Adhesion strength was measured further on 15 plaques affixed to porcine eyes (glued in five with intact conjunctiva, glued in five with removed conjunctiva, and sutured in five). Saline had no effect on the glue-plaque-eye complex, whereas the urokinase (0.38 mL ± 0.08 mL) easily dissolved the adhesion between the glue layer and surrounding tissues. The weight required to detach the plaques was 0.349 kg ± 0.173 kg for glued eyes with intact conjunctiva, 0.405 kg ± 0.083 kg for sutured eyes (P = .59), and 0.032 kg ± 0.004 kg for glued eyes without intact conjunctiva (P ≤ .015). The usage of the biological adhesive and dissolvent system was applicable for plaque surgery in an ex vivo animal model. Copyright 2016, SLACK Incorporated.

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

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

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

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.

A survey of the economics of materials processing in space. [accenting biomedical materials

NASA Technical Reports Server (NTRS)

Miller, B. P.

1975-01-01

A survey of the economics of space materials processing has been performed with the objectives of identifying those areas of space materials processing that give preliminary indication of significant economic potential, and to identify possible approaches to quantify the economic potential. It is concluded that limited economic studies have been performed to date, primarily in the area of the processing of inorganic materials, but that the economics of space processing of biological material has not received adequate attention. Specific studies are recommended to evaluate the economic impact of human lymphocyte subgroup separation on organ transplantation, and on the separation and concentration of urokinase producing cells.

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., works on an experiment found during the search for Columbia debris. Mike Casasanto, also with ITA, looks on. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., works on an experiment found during the search for Columbia debris. Mike Casasanto, also with ITA, looks on. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

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

[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.

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

KENNEDY SPACE CENTER, FLA. - Dr. Dennis Morrison, NASA Johnson Space Center, processes one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

NASA Image and Video Library

2003-05-07

KENNEDY SPACE CENTER, FLA. - Dr. Dennis Morrison, NASA Johnson Space Center, processes one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KENNEDY SPACE CENTER, FLA. - The crystals visible in this laboratory dish were part of an experiment carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

NASA Image and Video Library

2003-05-07

KENNEDY SPACE CENTER, FLA. - The crystals visible in this laboratory dish were part of an experiment carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., and Bob McLean, from the Southwest Texas State University, work on an experiment found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., and Bob McLean, from the Southwest Texas State University, work on an experiment found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto (foreground), Instrumentation Technology Associates, Inc., examines one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

NASA Image and Video Library

2003-05-07

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto (foreground), Instrumentation Technology Associates, Inc., examines one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KENNEDY SPACE CENTER, FLA. - Dr. Dennis Morrison, NASA Johnson Space Center, works with one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

NASA Image and Video Library

2003-05-07

KENNEDY SPACE CENTER, FLA. - Dr. Dennis Morrison, NASA Johnson Space Center, works with one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

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.

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

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

Preliminary benefit analysis of biological space processing

NASA Technical Reports Server (NTRS)

Perrine, J.

1976-01-01

The value of weightlessness in bioprocessing is assessed. The ecomonic benefits are assessed for space processing urokinase and human lymphocytes for treatment of end stage renal disease and thromboembolisms.

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., examines closely the container containing one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., examines closely the container containing one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

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.

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.

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.

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.

Application of the Ommaya Reservoir in Managing Ventricular Hemorrhage.

PubMed

Yang, Xi-Tao; Feng, Dong-Fu; Zhao, Liang; Sun, Zhao-Liang; Zhao, Gang

2016-05-01

Intraventricular hemorrhage (IVH) is associated with high morbidity and mortality. This study evaluated the safety and efficacy of the combined treatment of an Ommaya reservoir and conventional external ventricular drainage (EVD) using urokinase in the management of IVH. We performed a prospective controlled study. Sixty eligible patients with IVH received conventional EVD alone (group A) or combined EVD and Ommaya reservoir (group B) between January 2010 and January 2015. Clinical, cerebrospinal fluid, and radiographic data were used to assess clot clearance, clinical outcomes, and complications between the groups. There were no significant differences in gender, age, blood pressure, Glasgow Coma Scale, Graeb score, intracerebral hemorrhage volume on admission, and IVH volume before surgery between groups A and B (P > 0.05). The number of injections of urokinase (20,000 IU/dose) were significantly different in group B compared with group A (P < 0.05). Repeated computed tomography scans 3 days, 6 days, and 10 days after surgery revealed that clot clearance rates at each time point were significantly increased in group B compared with group A (P < 0.05). The conventional catheter-based EVD duration time was shortened to 5 (4-6) days in group B compared with 7 (5-9) days in group A (P < 0.05). The total drainage time was prolonged to 9 (8-11) days in group B compared with 7 (5-9) days in group A (P < 0.05). Ventriculitis was not significantly different between the 2 groups (P > 0.05). The hydrocephalus incidence and mortality revealed significant differences between the 2 groups (P < 0.05). The 30-day Glasgow Outcome Scale score was significantly increased in group B compared with group A (P < 0.05). The combined treatment approach of an Ommaya reservoir and EVD with intraventricular urokinase is safe and effective in patients with IVH. It increased clot clearance, shortened conventional catheter-based EVD duration, prolonged total drainage time, reduced the hydrocephalus incidence and mortality, and contributed to good clinical outcomes. The Ommaya reservoir provides a safe way to increase the injection times of urokinase, which accelerated clot resolution and did not increase the risk for ventriculitis infection. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, Instrumentation Technology Associates, Inc., studies one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

NASA Image and Video Library

2003-05-07

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, Instrumentation Technology Associates, Inc., studies one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

KENNEDY SPACE CENTER, FLA. - From left, Bob McLean, Southwest Texas State University, and Valerie Cassanto, Instrumentation Technology Associates, Inc., study one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

NASA Image and Video Library

2003-05-07

KENNEDY SPACE CENTER, FLA. - From left, Bob McLean, Southwest Texas State University, and Valerie Cassanto, Instrumentation Technology Associates, Inc., study one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KENNEDY SPACE CENTER, FLA. - From left, Valerie Cassanto, Instrumentation Technology Associates, Inc., and Dr. Dennis Morrison, NASA Johnson Space Center, analyze one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

NASA Image and Video Library

2003-05-07

KENNEDY SPACE CENTER, FLA. - From left, Valerie Cassanto, Instrumentation Technology Associates, Inc., and Dr. Dennis Morrison, NASA Johnson Space Center, analyze one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

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

Keeling, A. N.; Leong, S.; Logan, P. M.

Empyema and complicated pleural effusion represent common medical problems. Current treatment options are multiple. The purpose of this study was to access the outcome of image-guided, small-bore catheter drainage of empyema and effusion. We evaluated 93 small-bore catheters in 82 patients with pleural effusion (n = 30) or empyema (n = 52), over a 2-year period. Image guidance was with ultrasound (US; n = 56) and CT (n = 37). All patients were followed clinically, with catheter dwell times, catheter outcome, pleural fluid outcome, reinsertion rates, and need for urokinase or surgery recorded. Ninety-three small-bore chest drains (mean=10.2 Fr; range,more » 8.2-12.2 Fr) were inserted, with an average dwell time of 7.81 days for empyemas and 7.14 days for effusions (p > 0.05). Elective removal rates (73% empyema vs 86% effusions) and dislodgement rates (12% empyema vs 13% effusions) were similar for both groups. Eight percent of catheters became blocked and 17% necessitated reinsertion in empyemas, with no catheters blocked or requiring reinsertion in effusions (p < 0.05). Thirty-two patients (51%) required urokinase in the empyema group, versus 2 patients (6%) in the effusion group (p < 0.05). All treatment failures, requiring surgery, occurred in the empyema group (19%; n = 12; p < 0.05). In conclusion, noninfected pleural collections are adequately treated with small-bore catheters, however, empyemas have a failure rate of 19%. The threshold for using urokinase and larger-bore catheters should be low in empyema.« less

Empyema and effusion: outcome of image-guided small-bore catheter drainage.

PubMed

Keeling, A N; Leong, S; Logan, P M; Lee, M J

2008-01-01

Empyema and complicated pleural effusion represent common medical problems. Current treatment options are multiple. The purpose of this study was to access the outcome of image-guided, small-bore catheter drainage of empyema and effusion. We evaluated 93 small-bore catheters in 82 patients with pleural effusion (n = 30) or empyema (n = 52), over a 2-year period. Image guidance was with ultrasound (US; n = 56) and CT (n = 37). All patients were followed clinically, with catheter dwell times, catheter outcome, pleural fluid outcome, reinsertion rates, and need for urokinase or surgery recorded. Ninety-three small-bore chest drains (mean=10.2 Fr; range, 8.2-12.2 Fr) were inserted, with an average dwell time of 7.81 days for empyemas and 7.14 days for effusions (p > 0.05). Elective removal rates (73% empyema vs 86% effusions) and dislodgement rates (12% empyema vs 13% effusions) were similar for both groups. Eight percent of catheters became blocked and 17% necessitated reinsertion in empyemas, with no catheters blocked or requiring reinsertion in effusions (p < 0.05). Thirty-two patients (51%) required urokinase in the empyema group, versus 2 patients (6%) in the effusion group (p < 0.05). All treatment failures, requiring surgery, occurred in the empyema group (19%; n = 12; p < 0.05). In conclusion, noninfected pleural collections are adequately treated with small-bore catheters, however, empyemas have a failure rate of 19%. The threshold for using urokinase and larger-bore catheters should be low in empyema.

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

Hyperforin, a bio-active compound of St. John's Wort, is a new inhibitor of angiogenesis targeting several key steps of the process.

PubMed

Martínez-Poveda, Beatriz; Quesada, Ana R; Medina, Miguel Angel

2005-12-10

Hyperforin, a phloroglucinol derivative found in St. John's wort related mainly to its antidepressant effects, has been reported recently to induce apoptosis in tumour cells and to inhibit cancer invasion and metastasis. We show that hyperforin inhibits angiogenesis in vitro in bovine aortic endothelial cells and in vivo in the chorioallantoic membrane assay. In a variety of experimental systems representing the sequential events of the angiogenic process, hyperforin treatment of endothelial cells resulted in strong inhibitory effects. Hyperforin inhibited the growth of endothelial cells in culture. Capillary tube formation on Matrigel was abrogated completely by addition of hypeforin at the low micromolar range. Hyperforin also exhibited a clear inhibitory effect on the invasive capabilities of endothelial cells. Zymographic assays showed that hyperforin treatment produced a complete inhibition of urokinase and a remarkable inhibition of matrix metalloproteinase 2. Our data indicates that hyperforin is a compound that interferes with key events in angiogenesis, confirming the recent and growing evidence about a potential role of this compound in cancer and metastasis inhibition and making it a promising drug for further evaluation in the treatment of angiogenesis-related pathologies. Copyright 2005 Wiley-Liss, Inc

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.

KENNEDY SPACE CENTER, FLA. - Barry Perlman, Pembroke Pines Charter Middle School in Florida, prepares a computer to receive data from an experiment carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

NASA Image and Video Library

2003-05-07

KENNEDY SPACE CENTER, FLA. - Barry Perlman, Pembroke Pines Charter Middle School in Florida, prepares a computer to receive data from an experiment carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

KENNEDY SPACE CENTER, FLA. - From left, Bob McLean, Southwest Texas State University; Valerie Cassanto, Instrumentation Technology Associates, Inc.; and Dennis Morrison, NASA Johnson Space Center, process one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

NASA Image and Video Library

2003-05-07

KENNEDY SPACE CENTER, FLA. - From left, Bob McLean, Southwest Texas State University; Valerie Cassanto, Instrumentation Technology Associates, Inc.; and Dennis Morrison, NASA Johnson Space Center, process one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., and Bob McLean, from the Southwest Texas State University, transfer to a new container material from one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., and Bob McLean, from the Southwest Texas State University, transfer to a new container material from one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

Statistical Profiling of One Promiscuous Protein Binding Site: Illustrated by Urokinase Catalytic Domain.

PubMed

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

2017-10-01

While recent literature focuses on drug promiscuity, the characterization of promiscuous binding sites (ability to bind several ligands) remains to be explored. Here, we present a proteochemometric modeling approach to analyze diverse ligands and corresponding multiple binding sub-pockets associated with one promiscuous binding site to characterize protein-ligand recognition. We analyze both geometrical and physicochemical profile correspondences. This approach was applied to examine the well-studied druggable urokinase catalytic domain inhibitor binding site, which results in a large number of complex structures bound to various ligands. This approach emphasizes the importance of jointly characterizing pocket and ligand spaces to explore the impact of ligand diversity on sub-pocket properties and to establish their main profile correspondences. This work supports an interest in mining available 3D holo structures associated with a promiscuous binding site to explore its main protein-ligand recognition tendency. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Active Plasma Kallikrein Localizes to Mast Cells and Regulates Epithelial Cell Apoptosis, Adipocyte Differentiation, and Stromal Remodeling during Mammary Gland Involution*

PubMed Central

Lilla, Jennifer N.; Joshi, Ravi V.; Craik, Charles S.; Werb, Zena

2009-01-01

The plasminogen cascade of serine proteases directs both development and tumorigenesis in the mammary gland. Plasminogen can be activated to plasmin by urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasma kallikrein (PKal). The dominant plasminogen activator for mammary involution is PKal, a serine protease that participates in the contact activation system of blood coagulation. We observed that the prekallikrein gene (Klkb1) is expressed highly in the mammary gland during stromal remodeling periods including puberty and postlactational involution. We used a variant of ecotin (ecotin-PKal), a macromolecular inhibitor of serine proteases engineered to be highly specific for active PKal, to demonstrate that inhibition of PKal with ecotin-PKal delays alveolar apoptosis, adipocyte replenishment, and stromal remodeling in the involuting mammary gland, producing a phenotype resembling that resulting from plasminogen deficiency. Using biotinylated ecotin-PKal, we localized active PKal to connective tissue-type mast cells in the mammary gland. Taken together, these results implicate PKal as an effector of the plasminogen cascade during mammary development. PMID:19297327

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.

NASA Tech Briefs, April 2007

NASA Technical Reports Server (NTRS)

2007-01-01

Topics include: Wearable Environmental and Physiological Sensing Unit; Broadband Phase Retrieval for Image-Based Wavefront Sensing; Filter Function for Wavefront Sensing Over a Field of View; Iterative-Transform Phase Retrieval Using Adaptive Diversity; Wavefront Sensing With Switched Lenses for Defocus Diversity; Smooth Phase Interpolated Keying; Maintaining Stability During a Conducted-Ripple EMC Test; Photodiode Preamplifier for Laser Ranging With Weak Signals; Advanced High-Definition Video Cameras; Circuit for Full Charging of Series Lithium-Ion Cells; Analog Nonvolatile Computer Memory Circuits; JavaGenes Molecular Evolution; World Wind 3D Earth Viewing; Lithium Dinitramide as an Additive in Lithium Power Cells; Accounting for Uncertainties in Strengths of SiC MEMS Parts; Ion-Conducting Organic/Inorganic Polymers; MoO3 Cathodes for High-Temperature Lithium Thin-Film Cells; Counterrotating-Shoulder Mechanism for Friction Stir Welding; Strain Gauges Indicate Differential-CTE-Induced Failures; Antibodies Against Three Forms of Urokinase; Understanding and Counteracting Fatigue in Flight Crews; Active Correction of Aberrations of Low-Quality Telescope Optics; Dual-Beam Atom Laser Driven by Spinor Dynamics; Rugged, Tunable Extended-Cavity Diode Laser; Balloon for Long-Duration, High-Altitude Flight at Venus; and Wide-Temperature-Range Integrated Operational Amplifier.

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

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

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.

Intrapleural Fibrinolytic Therapy for Residual Coagulated Hemothorax After Lung Surgery.

PubMed

Huang, Dayu; Zhao, Deping; Zhou, Yiming; Liu, Hongchen; Chen, Xiaofeng

2016-05-01

Many studies have described the use of intrapleural fibrinolytics for the treatment of complex pleural processes and traumatic hemothorax, but data are scarce regarding their use for hemothorax after lung surgery. To evaluate the utility of intrapleural fibrinolytic therapy with urokinase for residual coagulated hemothorax (blood clot accumulation in the pleural cavity) after lung surgery. From July 2009 to November 2013, 46 patients (33 males; mean age, 56.9 ± 10.7 years) were treated with intrapleural urokinase (250,000 IU per dose) for residual hemothorax after lung surgery. Complete response was defined as clinical improvement with complete drainage of the retained collection shown by chest X-ray, and partial response as substantial resolution with minimal residual opacity (<25 % of the thorax). Follow-up was at least 30 days. The procedure was successful in 42 patients (91.3 %), with complete response observed in 35/46 patients (76.1 %) and partial response in 7/46 (15.2 %). These 42 patients did not require re-intervention for fluid accumulation in the pleural cavity. Treatment failed in 4 patients (8.7 %): one developed bronchopleural fistula that later resolved spontaneously and three (6.5 %) required thoracoscopic drainage for pleural cavity fluid accumulation and lung collapse. No patient required thoracotomy for total decortication. Intrapleural urokinase administration was not associated with serious adverse events, including bleeding complications or allergic reactions. Intrapleural fibrinolytic agents should be considered a useful therapeutic option for the treatment of postoperative residual hemothorax. This method appears to be safe and effective in >90 % of patients with postoperative hemothorax.

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

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.

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.

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.

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.

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.

Suppressive effects of a proton beam on tumor growth and lung metastasis through the inhibition of metastatic gene expression in 4T1 orthotopic breast cancer model.

PubMed

Kwon, Yun-Suk; Lee, Kyu-Shik; Chun, So-Young; Jang, Tae Jung; Nam, Kyung-Soo

2016-07-01

A proton beam is a next generation tool to treat intractable cancer. Although the therapeutic effects of a proton beam are well known, the effect on tumor metastasis is not fully described. Here, we investigated the effects of a proton beam on metastasis in highly invasive 4T1 murine breast cancer cells and their orthotopic breast cancer model. Cells were irradiated with 2, 4, 8 or 16 Gy proton beam, and changes in cell proliferation, survival, and migration were observed by MTT, colony forming and wound healing assays. 4T1 breast cancer cell-implanted BALB/c mice were established and the animals were randomly divided into 4 groups when tumor size reached 200 mm3. Breast tumors were selectively irradiated with 10, 20 or 30 Gy proton beam. Breast tumor sizes were measured twice a week, and breast tumor and lung tissues were pathologically observed. Metastasis-regulating gene expression was assessed with quantitative RT-PCR. A proton beam dose-dependently decreased cell proliferation, survival and migration in 4T1 murine breast cancer cells. Also, growth of breast tumors in the 4T1 orthotopic breast cancer model was significantly suppressed by proton beam irradiation without significant change of body weight. Furthermore, fewer tumor nodules metastasized from breast tumor into lung in mice irradiated with 30 Gy proton beam, but not with 10 and 20 Gy, than in control. We observed correspondingly lower expression levels of urokinase plasminogen activator (uPA), uPA receptor, cyclooxygenase (COX)-2, and vascular endothelial growth factor (VEGF), which are important factors in cancer metastasis, in breast tumor irradiated with 30 Gy proton beam. Proton beam irradiation did not affect expressions of matrix metalloproteinase (MMP)-9 and MMP-2. Taken together, the data suggest that, although proton beam therapy is an effective tool for breast cancer treatment, a suitable dose is necessary to prevent metastasis-linked relapse and poor prognosis.

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

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.

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.

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.

KSC-03pd1445

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - Dennis Morrison, senior biotech program scientist, talks to a reporter about an experiment recovered during the search for Columbia debris. He is the principle investigator on microencapsulation and urokinase crystal growth included in the Commercial ITA Biomedical Experiments payload on mission STS-107.

KSC-03pd1443

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - Dennis Morrison, senior biotech program scientist, talks to the media about an experiment recovered during the search for Columbia debris. He is the principle investigator on microencapsulation and urokinase crystal growth included in the Commercial ITA Biomedical Experiments payload on mission STS-107.

KSC-03pd1444

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - Dennis Morrison, senior biotech program scientist, talks to a reporter about an experiment recovered during the search for Columbia debris. He is the principle investigator on microencapsulation and urokinase crystal growth included in the Commercial ITA Biomedical Experiments payload on mission STS-107.

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

Gregorio, Miguel A. de; Ruiz, Carlos; Alfonso, Eduardo R.

Purpose: To assess the efficacy of intrapleural urokinase instillation through small-caliber catheters for the treatment of loculate and/or septate effusions. Methods: We inserted small-caliber catheters (8.2 Fr) in 102 patients with septate and/or loculate pleural effusions using ultrasonographic guidance. Urokinase (100,000 IU/2 hr, 3 times a day) was instilled through the catheter until the effusion resolved and D-dimer levels were <500 ng/ml. Patients were enrolled regardless of the etiology of the pleural effusion provided there were no contraindications for the use of urokinase. D-dimer levels were determined before and after treatment. Follow-up was performed by chest radiograph and sonography atmore » 1 day, 7 days, and every 30 days thereafter for 6 months. Results: Successful catheter placement was achieved in all cases. The mean time catheters stayed in place was 5.7 days and the mean dose of drug instilled was 690,000 IU. Pleural effusion drainage was complete at the first assessment in all patients. Failure of the treatment, with recurrent effusion at 30 days, occurred in six patients (5.8%). Complete resolution without sequelae was observed in 19 patients (19.6%). In 75 cases (73.5%) resolution was partial, with pleural thickening (>2 mm). Two patients died from unrelated causes within 30 days after catheter placement. Complications were seen in 13 patients (12.74%): hydropneumothorax, nine cases (8.82%); infection of the puncture point, three cases (2.94%); and adverse reaction, one case (0.98%). No further treatment was required. Conclusion: The use of intrapleural fibrinolytic agents delivered through small-caliber catheters for the treatment of loculate and/or septate pleural effusion is a simple, effective, minimally invasive and inexpensive procedure that can prevent sequelae and shorten drainage time.« less

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

De Gregorio, Miguel A.; Ruiz, Carlos; Alfonso, Eduardo R.

Purpose: To assess the efficacy of intrapleural urokinase instillation through smll-caliber catheters for the treatment of loculate and/or septate effusions.Methods: We inserted small-caliber catheters (8.2 Fr) in 102 patients with septate and/or loculate pleural effusions using ultrasonographic guidance. Urokinase (100,000 IU/2 hr, 3 times a day) was instilled through the catheter until the effusion resolved and D-dimer levels were <500 ng/ml. Patients were enrolled regardless of the etiology of the pleural effusion provided there were no contraindications for the use of urokinase. D-dimer levels were determined before and after treatment. Follow-up was performed by chest radiograph and sonography at 1more » day, 7 days, and every 30 days thereafter for 6 months.Results: Successful catheter placement was achieved in all cases. The mean time catheters stayed in place was 5.7 days and the mean dose of drug instilled was 690,000 IU. Pleural effusion drainage was complete at the first assessment in all patients. Failure of the treatment, with recurrent effusion at 30 days, occurred in six patients (5.8%). Complete resolution without sequelae was observed in 19 patients (19.6%). In 75 cases (73.5%) resolution was partial, with pleural thickening (>2 mm). Two patients died from unrelated causes within 30 days after catheter placement. Complications were seen in 13 patients (12.74%): hydropneumothorax, nine cases (8.82%); infection of the puncture point, three cases (2.94%); and adverse reaction, one case (0.98%). No further treatment was required.Conclusions: The use of intrapleural fibrinolytic agents delivered through small-caliber catheters for the treatment of loculate and/or septate pleural effusion is a simple, effective, minimally invasive and inexpensive procedure that can prevent sequelae and shorten drainage time.« less

Antithrombotic drug therapy for IgA nephropathy: a meta analysis of randomized controlled trials.

PubMed

Liu, Xiu-Juan; Geng, Yan-Qiu; Xin, Shao-Nan; Huang, Guo-Ming; Tu, Xiao-Wen; Ding, Zhong-Ru; Chen, Xiang-Mei

2011-01-01

Antithrombotic agents, including antiplatelet agents, anticoagulants and thrombolysis agents, have been widely used in the management of immunoglobulin A (IgA) nephropathy in Chinese and Japanese populations. To systematically evaluate the effects of antithrombotic agents for IgA nephropathy. Data sources consisted of MEDLINE, EMBASE, the Cochrane Library, Chinese Biomedical Literature Database (CBM), Chinese Science and Technology Periodicals Databases (CNKI) and Japana Centra Revuo Medicina (http://www.jamas.gr.jp) up to April 5, 2011. The quality of the studies was evaluated from the intention to treat analysis and allocation concealment, as well as by the Jadad method. Meta-analyses were performed on the outcomes of proteinuria and renal function. Six articles met the predetermined inclusion criteria. Antithrombotic agents showed statistically significant effects on proteinuria (p<0.0001) but not on the protection of renal function (p=0.07). The pooled risk ratio for proteinuria was 0.53, [95% confidence intervals (CI): 0.41-0.68; I(2)=0%] and for renal function it was 0.42 (95% CI 0.17-1.06; I(2)=72%). Subgroup analysis showed that dipyridamole was beneficial for proteinuria (p=0.0003) but had no significant effects on protecting renal function. Urokinase had statistically significant effects both on the reduction of proteinuria (p=0.0005) and protecting renal function (p<0.00001) when compared with the control group. Antithrombotic agents had statistically significant effects on the reduction of proteinuria but not on the protection of renal function in patients with IgAN. Urokinase had statistically significant effects both on the reduction of proteinuria and on protecting renal function. Urokinase was shown to be a promising medication and should be investigated further.

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

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

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

Selection of High-Affinity Peptidic Serine Protease Inhibitors with Increased Binding Entropy from a Back-Flip Library of Peptide-Protease Fusions.

PubMed

Sørensen, Hans Peter; Xu, Peng; Jiang, Longguang; Kromann-Hansen, Tobias; Jensen, Knud J; Huang, Mingdong; Andreasen, Peter A

2015-09-25

We have developed a new concept for designing peptidic protein modulators, by recombinantly fusing the peptidic modulator, with randomized residues, directly to the target protein via a linker and screening for internal modulation of the activity of the protein. We tested the feasibility of the concept by fusing a 10-residue-long, disulfide-bond-constrained inhibitory peptide, randomized in selected positions, to the catalytic domain of the serine protease murine urokinase-type plasminogen activator. High-affinity inhibitory peptide variants were identified as those that conferred to the fusion protease the lowest activity for substrate hydrolysis. The usefulness of the strategy was demonstrated by the selection of peptidic inhibitors of murine urokinase-type plasminogen activator with a low nanomolar affinity. The high affinity could not have been predicted by rational considerations, as the high affinity was associated with a loss of polar interactions and an increased binding entropy. Copyright © 2015 Elsevier Ltd. All rights reserved.

KENNEDY SPACE CENTER, FLA. - From left, Barry Perlman, Pembroke Pines Charter Middle School in Florida, and Valerie Cassanto, Instrumentation Technology Associates, Inc., analyze one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

NASA Image and Video Library

2003-05-07

KENNEDY SPACE CENTER, FLA. - From left, Barry Perlman, Pembroke Pines Charter Middle School in Florida, and Valerie Cassanto, Instrumentation Technology Associates, Inc., analyze one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

KENNEDY SPACE CENTER, FLA. - From left, Barry Perlman, Pembroke Pines Charter Middle School in Florida, and Valerie Cassanto, Instrumentation Technology Associates, Inc., process one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

NASA Image and Video Library

2003-05-07

KENNEDY SPACE CENTER, FLA. - From left, Barry Perlman, Pembroke Pines Charter Middle School in Florida, and Valerie Cassanto, Instrumentation Technology Associates, Inc., process one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

KENNEDY SPACE CENTER, FLA. - Barry Perlman, Pembroke Pines Charter Middle School in Florida, examines one of the experiments carried on mission STS-107 as Bob McLean, Southwest Texas State University, looks on. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

NASA Image and Video Library

2003-05-07

KENNEDY SPACE CENTER, FLA. - Barry Perlman, Pembroke Pines Charter Middle School in Florida, examines one of the experiments carried on mission STS-107 as Bob McLean, Southwest Texas State University, looks on. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

[Comparison of the intracranial pressure value in patients with hypertensive intracerebral hemorrhage treated with traditional craniotomy and puncture drainage].

PubMed

Song, Shao-jun; Fei, Zhou; Zhang, Xiang

2003-09-01

To compare the difference of intracranial pressure (ICP) in patients with hypertensive intracerebral hemorrhage (HICH) treated with two surgical procedures, traditional craniotomy and puncture drainage. One hundred and twelve cases with HICH were randomly divided into two groups. In one group, 60 patients were operated by traditional craniotomy and in another group, 52 cases by puncture drainage and urokinase treatment. In the meantime, ICP was monitored by placing catheter in lateral ventricle on the contralateral side of the hemorrhage. ICP values were recorded after operation at once, at 24 hours, 72 hours and 1 week. Although all the patients showed increased ICP, the increasing degree in patients treated with traditional craniotomy had lower ICP values (P<0.05 or P<0.01). Traditional craniotomy has advantages over puncture drainage for patients with HICH at least with respect to decreasing ICP.

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.

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.

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.

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.

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.

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.

KSC-03PD-1450

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto is one of the scientists recovering experiments found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KSC-03PD-1453

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., works on an experiment found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

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

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.

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

Hemostasis and malignancy.

PubMed

Francis, J L; Biggerstaff, J; Amirkhosravi, A

1998-01-01

There is considerable evidence that the hemostatic system is involved in the growth and spread of malignant disease. There is an increased incidence of thromboembolic disease in patients with cancers and hemostatic abnormalities are extremely common in such patients. Antihemostatic agents have been successfully used to treat a variety of experimental tumors, and several clinical trials in humans have been initiated. Although metastasis is undoubtedly multifactorial, intravascular coagulation activation and peritumor fibrin deposition seem to be important. The mechanisms by which hemostatic activation facilitates the malignant process remain to be completely elucidated. Of central importance may be the presence on malignant cells of tissue factor and urokinase receptor. Recent studies have suggested that these proteins, and others, may be involved at several stages of metastasis, including the key event of neovascularization. Tissue factor, the principal initiator of coagulation, may have additional roles, outside of fibrin formation, that are central to the biology of some solid tumors.

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

Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

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

Lund, Leif R; Romer, John; Thomasset, Nicole

Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when {beta}-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when {beta}-casein gene expression was being extinguished. Expression of sulfatedmore » glycoprotein-2 (SGP-2), interleukin-1{beta} converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin-1 and uPA were weakly induced, if at all, in hydrocortisonetreated mice. Furthermore, mRNA for membrane-type matrix metalloproteinase decreased after hydrocortisone treatment and paralleled the almost complete inhibition of activation of latent gelatinase A. Concomitantly, the gland filled with an overabundance of milk. Our data support the hypothesis that there are at least two distinct phases of involution: an initial phase, characterized by induction of the apoptosis-associated genes SGP-2 and ICE and apoptosis of fully differentiated mammary epithelial cells without visible degradation of the extracellular matrix, and a second phase, characterized by extracellular matrix remodeling and altered mesenchymal-epithelial interactions, followed by apoptosis of cells that are losing differentiated functions.« less

KENNEDY SPACE CENTER, FLA. - From left, Barry Perlman, Pembroke Pines Charter Middle School in Florida; Valerie Cassanto, Instrumentation Technology Associates, Inc.; and Dr. Dennis Morrison, NASA Johnson Space Center, process one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

NASA Image and Video Library

2003-05-07

KENNEDY SPACE CENTER, FLA. - From left, Barry Perlman, Pembroke Pines Charter Middle School in Florida; Valerie Cassanto, Instrumentation Technology Associates, Inc.; and Dr. Dennis Morrison, NASA Johnson Space Center, process one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

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.

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.

Association of late-onset Alzheimer disease with a genotype of PLAU, the gene encoding urokinase-type plasminogen activator on chromosome 10q22.2.

PubMed

Finckh, U; van Hadeln, K; Müller-Thomsen, T; Alberici, A; Binetti, G; Hock, C; Nitsch, R M; Stoppe, G; Reiss, J; Gal, A

2003-08-01

Urokinase-type plasminogen activator (uPA) converts plasminogen to plasmin. Plasmin is involved in processing of amyloid precursor protein and degrades secreted and aggregated amyloid-beta, a hallmark of Alzheimer disease (AD). PLAU, the gene encoding uPA, maps to chromosome 10q22.2 between two regions showing linkage to late-onset AD (LOAD). We genotyped a frequent C/T single nucleotide polymorphism in codon 141 of PLAU (P141L) in 347 patients with LOAD and 291 control subjects. LOAD was associated with homozygous C/C PLAU genotype in the whole sample (chi2=15.7, P=0.00039, df 2), as well as in all sub-samples stratified by gender or APOE epsilon4 carrier status (chi2> or = 6.84, P< or =0.033, df 2). Odds ratio for LOAD due to homozygosity C/C was 1.89 (95% confidence interval 1.37-2.61). PLAU is a promising new candidate gene for LOAD, with allele C (P141) being a recessive risk allele or allele T (L141) conferring protection.

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.

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.

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.

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.

Early endovascular treatment of superior mesenteric occlusion secondary to thromboemboli.

PubMed

Jia, Z; Jiang, G; Tian, F; Zhao, J; Li, S; Wang, K; Wang, Y; Jiang, L; Wang, W

2014-02-01

To evaluate our early experience with endovascular revascularization in patients with acute thromboembolic occlusion of the superior mesenteric artery (SMA). A retrospective review was conducted of all patients who underwent endovascular revascularization for acute thromboembolic SMA occlusion from May 2005 to May 2012. Endovascular revascularization was performed using aspiration, intra-arterial thrombolysis, and adjunctive stent-placement techniques. Laparotomy was performed if the patient developed clinical signs of advanced bowel ischemia after endovascular procedure. Twenty-one patients underwent endovascular revascularization for acute thromboembolic SMA occlusion. All presented with acute-onset abdominal pain. Three patients had rebound tenderness before the procedure. Computed tomography angiography revealed complete occlusion in seven cases and incomplete occlusion in 14 cases, with no evidence of free gas or bowel necrosis. The median duration from onset of symptoms to revascularization was 8.7 ± 4.1 hours (range, 2-18 hours). Completely successful endovascular revascularization occurred in six cases (aspiration alone, 3 cases; combined aspiration and urokinase, 3 cases); partial success was achieved in 15 cases (aspiration alone, 4 cases; combined aspiration and urokinase, 10 cases; and combined aspiration, urokinase, and stent placement, 1 case). Laparotomy was required in five patients, all of whom had SMA main trunk complete occlusion and required small bowel resection. The 30-day mortality for all patients was 9.5%. During a median follow-up of 26 months, 15 patients remained asymptomatic, three patients reported occasional abdominal pain, and one patient had temporary short-bowel syndrome. Percutaneous revascularization is a promising alternative to surgery for acute SMA occlusion in selected patients who have no signs of advanced bowel ischemia. Early diagnosis followed by prompt endovascular intervention with close postprocedural monitoring is key. Laparotomy is indicated in patients who develop new or worsening signs of peritonism after endovascular procedure, particularly in those who had complete occlusion of the main trunk of the SMA. Copyright © 2013 European Society for Vascular Surgery. All rights reserved.

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

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.

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

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.

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.

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

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.

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.

[An efficient strategy to decrease the central venous catheter-related adverse events rate in haemodialysis patients].

PubMed

Jean, Guillaume; Vanel, Thierry; Bresson, Eric; Terrat, Jean-Claude; Hurot, Jean-Marc; Lorriaux, Christie; Mayor, Brice; Chazot, Charles

2009-07-01

Catheter-related adverse events (CAE) remain a major cause of mortality and morbidity. We aimed to compare the CAE prevalence and adverse events rate at 10 years interval in one centre using different devices, dressing procedures. We compared two periods, from 1994 to 1997 (period 1) and from 2004 to 2007 (period 2). We recorded all prevalent tunnelled CAE and their related adverse event rate: catheter-related bacteraemia (CRB), catheter local infection (CLI), catheter dysfunction leading to CAE exchange, thrombolytic use and spontaneous pulling up. In period 1, PermCath catheter (Quinton, N=63) and TwinCath catheter (MedComp, N=76) were used in 95 HD. BioFlex catheter (N=52) and ASPC split catheter (MedComp, N=52) were used in 72 HD in period 2. In period 1, we performed catheter dressing using povidone iodine versus alcoholic chlorexidine in period 2. Between period 1 and period 2, the CAE prevalence decreased from 15-18% to 9-6%, CRB from 1.1 to 0.23/1000 day-catheter (p<0.001), CLI from 1.1 to 0.28/1000 day-catheter (p<0.001), definitive dysfunction from 12 to 1.2% (p<0.001) and CAE pulling up from 4 to 0%. The annual urokinase consumption decreased from three to one unit per CAE. This study shows the dramatic decrease in CAE prevalence (-50%) and related-adverse events (approximately -200%) since 10 years. Switching povidone iodine to chlorexidine and using more recent catheter devices appear very efficient in decreasing catheter-related adverse events.

KSC-03PD-1455

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., works on an experiment found during the search for Columbia debris. Mike Casasanto, also with ITA, looks on. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

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.

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.

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

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

[Analysis of salivary protease spectrum in chronic periodontitis].

PubMed

Qian, Li; Xuedong, Zhou; Yaping, Fan; Tengyu, Yang; Songtao, Wu; Yu, Yu; Jiao, Chen; Ping, Zhang; Yun, Feng

2017-02-01

This study aimed to investigate the difference in salivary protease expression in patients with chronic periodontitis and normal individuals. The stimulating saliva in patients with chronic periodontitis and normal individuals were collected. Protein chip technology was adapted to analyze salivary protease spectrum. Among the 34 proteases in the chip, disintegrin and metalloproteinase (ADAM)8, matrix metalloproteinase (MMP)-8, MMP-12, neprilysin/CD10, and uridylyl phosphate adenosine/urokinase showed a significantly increased concentration in the saliva of chronic periodontitis patients compared with those in the saliva of normal individuals (P<0.01). By contrast, the concentrations of ADAM9, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)1, ADAMTS13, cathepsin B, E, L, V, X/Z/P, kallikrein 6, 7, 11, 13, MMP-9, proteinase 3, presenilin-1, and proprotein convertase 9 sharply decreased (P<0.05). The results demonstrated that protease spectrum in the saliva of chronic periodontitis patients and normal individuals significantly differed. Analysis of salivary protease spectrum is a potential clinical method to examine, diagnose, and monitor chronic periodontitis.

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.

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

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

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

Visible Thrombolysis Acceleration of a Nanomachine Powered by Light-Driving F0F1-ATPase Motor

NASA Astrophysics Data System (ADS)

Duan, Xiaoxia; Liu, Lifeng; Jiang, Weijian; Yue, Jiachang

2015-05-01

We report on thrombolysis acceleration of a nanomachine powered by light-driving δ-subunit-free F0F1-ATPase motor. It is composed of a mechanical device, locating device, energy storage device, and propeller. The rotory δ-subunit-free F0F1-ATPase motor acts as a mechanical device, which was obtained by reconstructing an original chromatophore extracted from Rhodospirillum rubrum. We found that the bioactivity of the F0F1-ATPase motor improved greatly after reconstruction. The zeta potential of the nanomachine is about -23.4 mV. Cytotoxicity induced by the nanomachine was measured using cell counting kit (CCK)-8 assay. The A549 cells incubated with different fractional concentrations of the nanomachine within 48 h did not show obvious cytotoxicity. The locating device helps the nanomachine bind to the thrombi. Energy was easily stored by exposing the nanomachine to 600-nm-wavelength irradiation, which promoted activity of the motor. The rotation of the long propeller accelerated thrombolysis of a blood clot in vitro in the presence of urokinase (UK). This result was based on visual inspection and confirmed by a series of tests.

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.

KSC-03PD-1463

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Dr. Dennis Morrison, NASA Johnson Space Center, works with one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KSC-03PD-1460

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Dr. Dennis Morrison, NASA Johnson Space Center, processes one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KSC-03PD-1461

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto (foreground), Instrumentation Technology Associates, Inc., examines one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KSC-03PD-1471

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - The crystals visible in this laboratory dish were part of an experiment carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KSC-03PD-1454

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., and Bob McLean, from the Southwest Texas State University, work on an experiment found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

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

The effect of peripherally inserted central catheter (PICC) valve technology on catheter occlusion rates--the 'ELeCTRiC' study.

PubMed

Johnston, Andrew J; Streater, Carmel T; Noorani, Remy; Crofts, Joanne L; Del Mundo, Aldwin B; Parker, Richard A

2012-01-01

Peripherally Inserted Central Catheters (PICCs) are increasingly being used to provide short to medium-term central venous access. The current study was designed to test the hypothesis that PICC valve technology does not influence PICC occlusion rates. Intensive care unit (ICU) patients who required a PICC were randomized to one of three types of dual lumen PICC (open ended non-valved, Groshong valve, PASV valve). PICC occlusions were recorded and managed with a protocol that used urokinase. A total of 102 patients were recruited to the study. The overall risk of occlusion per catheter was 35% (95% CI 26% to 44%). The overall rate of occlusion was 76 occlusions per 1000 catheter days (95% CI 61 to 95). Presence or type of valve did not significantly influence this rate (open-ended non-valved PICC 38% of catheters, 79 occlusions per 1000 catheter days; Groshong 38% of catheters, 60 occlusions per 1000 catheter days; PASV 27% of catheters, 99 occlusions per 1000 catheter days). The dose of urokinase required to treat PICC occlusions did not significantly differ between PICC types. Valved PICCs do not appear to influence PICC occlusion rates.

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

Carbon-ion radiation enhances migration ability and invasiveness of the pancreatic cancer cell, PANC-1, in vitro.

PubMed

Fujita, Mayumi; Otsuka, Yoshimi; Imadome, Kaori; Endo, Satoshi; Yamada, Shigeru; Imai, Takashi

2012-04-01

Pancreatic cancer is an aggressive disease that responds poorly to conventional photon radiotherapy. Carbon-ion (C-ion) radiation has advantages compared with conventional radiotherapy, because it enables more accurate dose distribution and more efficient tumor cell killing. To elucidate the effects of local radiotherapy on the characteristics of metastatic tumors, it is necessary to understand the nature of motility in irradiated tumor cells; this will, in turn, facilitate the development of effective strategies to counter tumor cell motility, which can be used in combination with radiotherapy. The aim of the present study was to examine the invasiveness of pancreatic cancer cells exposed to C-ion irradiation. We found that C-ion irradiation suppressed the migration of MIAPaCa-2, BxPC-3 and AsPC-1; diminished the invasiveness of MIAPaCa-2; and tended to reduce the invasion of BxPC-3 and AsPC-1. However, C-ion irradiation increased the invasiveness of PANC-1 through the activation of plasmin and urokinase-type plasiminogen activator. Administration of serine protease inhibitor (SerPI) alone failed to reduce C-ion-induced PANC-1 invasiveness, whereas the combination of SerPI and Rho-associated coiled-coil forming protein kinase (ROCK) inhibitor suppressed it. Furthermore, PANC-1 showed mesenchymal-amoeboid transition when we treated with SerPI alone. In conclusion, C-ion irradiation is effective in suppressing the invasive potential of several pancreatic tumor cell lines, but not PANC-1; this is the first study showing that C-ion irradiation induces the invasive potential of a tumor cell line. Further in vivo studies are required to examine the therapeutic effectiveness of radiotherapy combined with inhibitors of both mesenchymal and amoeboid modes of tumor cell motility. © 2011 Japanese Cancer Association.

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

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.

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.

KSC-03PD-1456

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., examines closely the container containing one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

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

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

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

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

Tumor Penetrating Theranostic Nanoparticles for Enhancement of Targeted and Image-guided Drug Delivery into Peritoneal Tumors following Intraperitoneal Delivery.

PubMed

Gao, Ning; Bozeman, Erica N; Qian, Weiping; Wang, Liya; Chen, Hongyu; Lipowska, Malgorzata; Staley, Charles A; Wang, Y Andrew; Mao, Hui; Yang, Lily

2017-01-01

The major obstacles in intraperitoneal (i.p.) chemotherapy of peritoneal tumors are fast absorption of drugs into the blood circulation, local and systemic toxicities, inadequate drug penetration into large tumors, and drug resistance. Targeted theranostic nanoparticles offer an opportunity to enhance the efficacy of i.p. therapy by increasing intratumoral drug delivery to overcome resistance, mediating image-guided drug delivery, and reducing systemic toxicity. Herein we report that i.p. delivery of urokinase plasminogen activator receptor (uPAR) targeted magnetic iron oxide nanoparticles (IONPs) led to intratumoral accumulation of 17% of total injected nanoparticles in an orthotopic mouse pancreatic cancer model, which was three-fold higher compared with intravenous delivery. Targeted delivery of near infrared dye labeled IONPs into orthotopic tumors could be detected by non-invasive optical and magnetic resonance imaging. Histological analysis revealed that a high level of uPAR targeted, PEGylated IONPs efficiently penetrated into both the peripheral and central tumor areas in the primary tumor as well as peritoneal metastatic tumor. Improved theranostic IONP delivery into the tumor center was not mediated by nonspecific macrophage uptake and was independent from tumor blood vessel locations. Importantly, i.p. delivery of uPAR targeted theranostic IONPs carrying chemotherapeutics, cisplatin or doxorubicin, significantly inhibited the growth of pancreatic tumors without apparent systemic toxicity. The levels of proliferating tumor cells and tumor vessels in tumors treated with the above theranostic IONPs were also markedly decreased. The detection of strong optical signals in residual tumors following i.p. therapy suggested the feasibility of image-guided surgery to remove drug-resistant tumors. Therefore, our results support the translational development of i.p. delivery of uPAR-targeted theranostic IONPs for image-guided treatment of peritoneal tumors.

KSC-03pd1452

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto and Bob McLean talk to a reporter about experiments found during the search for Columbia debris. Cassanto is with Instrumentation Technology Associates Inc. and McLean is with the Southwest Texas State University. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

Superior vena cava syndrome with central venous catheter for chemotherapy treated successfully with fibrinolysis.

PubMed

Guijarro Escribano, J F; Antón, R F; Colmenarejo Rubio, A; Sáenz Cascos, L; Sainz González, F; Alguacil Rodríguez, R

2007-03-01

Recently, there has been an increase in the number of cases of superior vena cava (SVC) syndrome associated with chronic indwelling central venous catheters. Fibrinolytic therapy and endovascular treatment are currently achieving good results. We present a case history of a patient with SVC with a catheter used for chemotherapy, which was successfully treated with catheter-directed (intraclot) infusion thrombolytic therapy with urokinase.

KSC-03PD-1467

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, Instrumentation Technology Associates, Inc., studies one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

KSC-03PD-1470

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - From left, Bob McLean, Southwest Texas State University, and Valerie Cassanto, Instrumentation Technology Associates, Inc., study one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KSC-03PD-1452

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto and Bob McLean talk to a reporter about experiments found during the search for Columbia debris. Cassanto is with Instrumentation Technology Associates Inc. and McLean is with the Southwest Texas State University. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KSC-03PD-1462

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - From left, Valerie Cassanto, Instrumentation Technology Associates, Inc., and Dr. Dennis Morrison, NASA Johnson Space Center, analyze one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

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

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.

Mechanisms of Severe Acute Respiratory Syndrome Coronavirus-Induced Acute Lung Injury

PubMed Central

Gralinski, Lisa E.; Bankhead, Armand; Jeng, Sophia; Menachery, Vineet D.; Proll, Sean; Belisle, Sarah E.; Matzke, Melissa; Webb-Robertson, Bobbie-Jo M.; Luna, Maria L.; Shukla, Anil K.; Ferris, Martin T.; Bolles, Meagan; Chang, Jean; Aicher, Lauri; Waters, Katrina M.; Smith, Richard D.; Metz, Thomas O.; Law, G. Lynn; Katze, Michael G.; McWeeney, Shannon; Baric, Ralph S.

2013-01-01

ABSTRACT Systems biology offers considerable promise in uncovering novel pathways by which viruses and other microbial pathogens interact with host signaling and expression networks to mediate disease severity. In this study, we have developed an unbiased modeling approach to identify new pathways and network connections mediating acute lung injury, using severe acute respiratory syndrome coronavirus (SARS-CoV) as a model pathogen. We utilized a time course of matched virologic, pathological, and transcriptomic data within a novel methodological framework that can detect pathway enrichment among key highly connected network genes. This unbiased approach produced a high-priority list of 4 genes in one pathway out of over 3,500 genes that were differentially expressed following SARS-CoV infection. With these data, we predicted that the urokinase and other wound repair pathways would regulate lethal versus sublethal disease following SARS-CoV infection in mice. We validated the importance of the urokinase pathway for SARS-CoV disease severity using genetically defined knockout mice, proteomic correlates of pathway activation, and pathological disease severity. The results of these studies demonstrate that a fine balance exists between host coagulation and fibrinolysin pathways regulating pathological disease outcomes, including diffuse alveolar damage and acute lung injury, following infection with highly pathogenic respiratory viruses, such as SARS-CoV. PMID:23919993

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

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.

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.

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

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

KSC-03PD-1459

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - From left, Bob McLean, Southwest Texas State University; Valerie Cassanto, Instrumentation Technology Associates, Inc.; and Dennis Morrison, NASA Johnson Space Center, process one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

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.

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.

Carprofen simultaneously reduces progression of morphological changes in cartilage and subchondral bone in experimental dog osteoarthritis.

PubMed

Pelletier, J P; Lajeunesse, D; Jovanovic, D V; Lascau-Coman, V; Jolicoeur, F C; Hilal, G; Fernandes, J C; Martel-Pelletier, J

2000-12-01

To examine the effect of a nonsteroidal antiinflammatory drug, carprofen, on the structure and metabolism of cartilage and subchondral bone in the experimental osteoarthritic (OA) canine model. Experimental Groups 1 and 2 received a sectioning of the anterior cruciate ligament (ACL) of the right stifle joint, and were administered carprofen (2.2 and 4.4 mg/kg/twice daily/po, respectively) for 8 weeks beginning 4 weeks postsurgery. Group 3 received ACL sectioning and no treatment. Group 4 was composed of unoperated normal dogs. Cartilage macroscopic lesions were assessed, and their histological severity was graded. Specimens of subchondral bones were fixed, decalcified, and stained with hematoxylin/eosin. The level of metalloprotease (MMP) activity in cartilage was measured. Osteoblast cells were prepared from the subchondral bone. The level of synthesis of osteoblast biomarkers (osteocalcin, alkaline phosphatase), as well as urokinase plasminogen activator (uPA) activity and insulin-like growth factor (IGF-1) in the culture medium, was estimated. Carprofen treatment decreased the width of osteophytes (p < 0.01), the size of cartilage lesions, and the histologic severity of cartilage lesions (p < 0.008). There was no difference in the levels of MMP activity in cartilage between OA and carprofen treated groups. In OA dogs, the subchondral bone plate was thinner and was the site of an extensive remodeling process with numerous lacunae. Dogs treated with carprofen showed a marked decrease in the remodeling activity with normal plate thickness, and subchondral bone morphology resembling that of normal dogs. Osteoblasts from untreated OA dogs showed slightly higher alkaline phosphatase activities and osteocalcin release that reverted back to normal upon carprofen treatment. Moreover, uPA activity and IGF-1 levels were increased in OA dogs and were significantly reduced in carprofen treated dogs. Under therapeutic conditions, treatment with carprofen could reduce the progression of early structural changes in experimental OA. Carprofen treatment also delays and/or prevents the abnormal metabolism of subchondral osteoblasts in this model. The hypothesis of a possible link between the protective effect of carprofen and its effect on subchondral bone is of interest in the context of therapeutic intervention.

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

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

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

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

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

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.

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

RNA Regulation by Estrogen

DTIC Science & Technology

2011-08-01

activator, urokinase 6.06 207403_at IRS4 insulin receptor substrate 4 6.06 208487_at lmx1b LIM homeobox transcription factor 1, beta 6.06 206051_at...3.48 216533_at pccA propionyl Coenzyme A carboxylase, alpha 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

KSC-03pd1448

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - Pictured is one of the microcapsules removed from the Commercial ITA Biomedical Experiments payload recovered during the search for Columbia debris. The drug delivery system and spaceflight hardware was developed jointly by JSC, the Institute for Research Inc. and Instrumentation Technology Associates Inc. to conduct microencapsulation experiments under microgravity conditions. This microcapsule contains an antibiotic for treating deep resistant pulmonary infections. Dr. Dennis Morrison, senior biotech project scientist, is principle investigator on microencapsulation and urokinase crystal growth.

KSC-03pd1447

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - Pictured is one of the microcapsules removed from the Commercial ITA Biomedical Experiments payload recovered during the search for Columbia debris. The drug delivery system and spaceflight hardware was developed jointly by JSC, the Institute for Research Inc. and Instrumentation Technology Associates Inc. to conduct microencapsulation experiments under microgravity conditions. This microcapsule contains an antibiotic for treating deep resistant pulmonary infections. Dr. Dennis Morrison, senior biotech project scientist, is principle investigator on microencapsulation and urokinase crystal growth.

KSC-03pd1449

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - Pictured is one of the microcapsules removed from the Commercial ITA Biomedical Experiments payload recovered during the search for Columbia debris. The drug delivery system and spaceflight hardware was developed jointly by JSC, the Institute for Research Inc. and Instrumentation Technology Associates Inc. to conduct microencapsulation experiments under microgravity conditions. This microcapsule contains an antibiotic for treating deep resistant pulmonary infections. Dr. Dennis Morrison, senior biotech project scientist, is principle investigator on microencapsulation and urokinase crystal growth.

KSC-03PD-1449

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Pictured is one of the microcapsules removed from the Commercial ITA Biomedical Experiments payload recovered during the search for Columbia debris. The drug delivery system and spaceflight hardware was developed jointly by JSC, the Institute for Research Inc. and Instrumentation Technology Associates Inc. to conduct microencapsulation experiments under microgravity conditions. This microcapsule contains an antibiotic for treating deep resistant pulmonary infections. Dr. Dennis Morrison, senior biotech project scientist, is principle investigator on microencapsulation and urokinase crystal growth.

KSC-03PD-1447

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Pictured is one of the microcapsules removed from the Commercial ITA Biomedical Experiments payload recovered during the search for Columbia debris. The drug delivery system and spaceflight hardware was developed jointly by JSC, the Institute for Research Inc. and Instrumentation Technology Associates Inc. to conduct microencapsulation experiments under microgravity conditions. This microcapsule contains an antibiotic for treating deep resistant pulmonary infections. Dr. Dennis Morrison, senior biotech project scientist, is principle investigator on microencapsulation and urokinase crystal growth.

KSC-03PD-1448

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Pictured is one of the microcapsules removed from the Commercial ITA Biomedical Experiments payload recovered during the search for Columbia debris. The drug delivery system and spaceflight hardware was developed jointly by JSC, the Institute for Research Inc. and Instrumentation Technology Associates Inc. to conduct microencapsulation experiments under microgravity conditions. This microcapsule contains an antibiotic for treating deep resistant pulmonary infections. Dr. Dennis Morrison, senior biotech project scientist, is principle investigator on microencapsulation and urokinase crystal growth.

KSC-03PD-1457

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Valerie Cassanto, with Instrumentation Technology Associates, Inc., and Bob McLean, from the Southwest Texas State University, transfer to a new container material from one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation.

KSC-03PD-1472

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Barry Perlman, Pembroke Pines Charter Middle School in Florida, prepares a computer to receive data from an experiment carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

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.

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.

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.

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

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.

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.

Research in Biological and Medical Sciences, Including Biochemistry, Communicable Disease and Immunology, Internal Medicine, Nuclear Medicine, Physiology, Psychiatry, Surgery and Veterinary Medicine. Volume 2

DTIC Science & Technology

1975-07-01

hepatitis. In addition, fat vacuoles were observed in one specimen. The specimens from the other two men showed mild hepatocellular unrest and mild fatty...right heart pressures in patients with pulmonary embolism . These clinical observations suggest that urokinase has vaso- motor effects which have not...180: 236-242, 1974. 29. Spence, M. R., and Mason, K. G.: Experimental amniotic fluid embolism in rabbits. Am. J. Obst. Gynec. 119: 1073-1078, 1974

Successful Intra-Arterial Thrombolysis for Acute Ischemic Stroke in the Immediate Postpartum Period: Case Report

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

Mendez, Jose C.; Masjuan, J.; Garcia, N.

2008-01-15

Stroke in pregnancy and the puerperium is a rare but potentially devastating event. We present the case of a previously healthy woman who underwent a cesarean delivery and experienced a middle cerebral artery thrombosis in the immediate postpartum period that was subsequently lysed with intra-arterial urokinase. The patient made a complete neurologic recovery. To the best of our knowledge, this is the first reported case of successful intra-arterial thrombolysis for ischemic stroke in the postpartum period.

KSC-03PD-1464

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - From left, Barry Perlman, Pembroke Pines Charter Middle School in Florida, and Valerie Cassanto, Instrumentation Technology Associates, Inc., process one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

KSC-03PD-1465

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - From left, Barry Perlman, Pembroke Pines Charter Middle School in Florida, and Valerie Cassanto, Instrumentation Technology Associates, Inc., process one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

KSC-03PD-1466

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - From left, Barry Perlman, Pembroke Pines Charter Middle School in Florida, and Valerie Cassanto, Instrumentation Technology Associates, Inc., analyze one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

KSC-03PD-1468

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - Barry Perlman, Pembroke Pines Charter Middle School in Florida, examines one of the experiments carried on mission STS-107 as Bob McLean, Southwest Texas State University, looks on. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

Suppression of growth and invasive behavior of human prostate cancer cells by ProstaCaid™: mechanism of activity.

PubMed

Jiang, Jiahua; Eliaz, Isaac; Sliva, Daniel

2011-06-01

Since the use of dietary supplements as alternative treatments or adjuvant therapies in cancer treatment is growing, a scientific verification of their biological activity and the detailed mechanisms of their action are necessary for the acceptance of dietary supplements in conventional cancer treatments. In the present study we have evaluated the anti-cancer effects of dietary supplement ProstaCaid™ (PC) which contains mycelium from medicinal mushrooms (Ganoderma lucidum, Coriolus versicolor, Phellinus linteus), saw palmetto berry, pomegranate, pumpkin seed, green tea [40% epigallocatechin-3-gallate (EGCG)], Japanese knotweed (50% resveratrol), extracts of turmeric root (BCM-95®), grape skin, pygeum bark, sarsaparilla root, Scutellaria barbata, eleuthero root, Job's tears, astragalus root, skullcap, dandelion, coptis root, broccoli, and stinging nettle, with purified vitamin C, vitamin D3, selenium, quercetin, citrus bioflavonoid complex, β sitosterolzinc, lycopene, α lipoic acid, boron, berberine and 3.3'-diinodolymethane (DIM). We show that PC treatment resulted in the inhibition of cell proliferation of the highly invasive human hormone refractory (independent) PC-3 prostate cancer cells in a dose- and time-dependent manner with IC50 56.0, 45.6 and 39.0 µg/ml for 24, 48 and 72 h, respectively. DNA-microarray analysis demonstrated that PC inhibits proliferation through the modulation of expression of CCND1, CDK4, CDKN1A, E2F1, MAPK6 and PCNA genes. In addition, PC also suppresses metastatic behavior of PC-3 by the inhibition of cell adhesion, cell migration and cell invasion, which was associated with the down-regulation of expression of CAV1, IGF2, NR2F1, and PLAU genes and suppressed secretion of the urokinase plasminogen activator (uPA) from PC-3 cells. In conclusion, the dietary supplement PC is a promising natural complex with the potency to inhibit invasive human prostate cancer.

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.

Regulation of DNA repair mechanism in human glioma xenograft cells both in vitro and in vivo in nude mice.

PubMed

Ponnala, Shivani; Veeravalli, Krishna Kumar; Chetty, Chandramu; Dinh, Dzung H; Rao, Jasti S

2011-01-01

Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells. Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls. Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point.

Regulation of DNA Repair Mechanism in Human Glioma Xenograft Cells both In Vitro and In Vivo in Nude Mice

PubMed Central

Ponnala, Shivani; Veeravalli, Krishna Kumar; Chetty, Chandramu; Dinh, Dzung H.; Rao, Jasti S.

2011-01-01

Background Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells. Methodology/Principal Findings Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls. Conclusion/Significance Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point. PMID:22022560

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

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.

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.

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

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

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.

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.

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.

Matriptase activation connects tissue factor-dependent coagulation initiation to epithelial proteolysis and signaling.

PubMed

Le Gall, Sylvain M; Szabo, Roman; Lee, Melody; Kirchhofer, Daniel; Craik, Charles S; Bugge, Thomas H; Camerer, Eric

2016-06-23

The coagulation cascade is designed to sense tissue injury by physical separation of the membrane-anchored cofactor tissue factor (TF) from inactive precursors of coagulation proteases circulating in plasma. Once TF on epithelial and other extravascular cells is exposed to plasma, sequential activation of coagulation proteases coordinates hemostasis and contributes to host defense and tissue repair. Membrane-anchored serine proteases (MASPs) play critical roles in the development and homeostasis of epithelial barrier tissues; how MASPs are activated in mature epithelia is unknown. We here report that proteases of the extrinsic pathway of blood coagulation transactivate the MASP matriptase, thus connecting coagulation initiation to epithelial proteolysis and signaling. Exposure of TF-expressing cells to factors (F) VIIa and Xa triggered the conversion of latent pro-matriptase to an active protease, which in turn cleaved the pericellular substrates protease-activated receptor-2 (PAR2) and pro-urokinase. An activation pathway-selective PAR2 mutant resistant to direct cleavage by TF:FVIIa and FXa was activated by these proteases when cells co-expressed pro-matriptase, and matriptase transactivation was necessary for efficient cleavage and activation of wild-type PAR2 by physiological concentrations of TF:FVIIa and FXa. The coagulation initiation complex induced rapid and prolonged enhancement of the barrier function of epithelial monolayers that was dependent on matriptase transactivation and PAR2 signaling. These observations suggest that the coagulation cascade engages matriptase to help coordinate epithelial defense and repair programs after injury or infection, and that matriptase may contribute to TF-driven pathogenesis in cancer and inflammation.

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.

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.

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.

KSC-03pd1446

NASA Image and Video Library

2003-05-06

KENNEDY SPACE CENTER, FLA. - The apparatus shown was designed to hold microcapsules for research on mission STS-107. It is one over several included in the Commercial ITA Biomedical Experiments payload. The box was recently recovered during the search for Columbia debris. The drug delivery system and spaceflight hardware was developed jointly by JSC, the Institute for Research Inc. and Instrumentation Technology Associates Inc. to conduct microencapsulation experiments under microgravity conditions. This microcapsule contains an antibiotic for treating deep resistant pulmonary infections. Dr. Dennis Morrison, senior biotech project scientist, is principle investigator on microencapsulation and urokinase crystal growth.

KSC-03PD-1446

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - The apparatus shown was designed to hold microcapsules for research on mission STS-107. It is one over several included in the Commercial ITA Biomedical Experiments payload. The box was recently recovered during the search for Columbia debris. The drug delivery system and spaceflight hardware was developed jointly by JSC, the Institute for Research Inc. and Instrumentation Technology Associates Inc. to conduct microencapsulation experiments under microgravity conditions. This microcapsule contains an antibiotic for treating deep resistant pulmonary infections. Dr. Dennis Morrison, senior biotech project scientist, is principle investigator on microencapsulation and urokinase crystal growth.

KSC-03PD-1469

NASA Technical Reports Server (NTRS)

2003-01-01

KENNEDY SPACE CENTER, FLA. - From left, Barry Perlman, Pembroke Pines Charter Middle School in Florida; Valerie Cassanto, Instrumentation Technology Associates, Inc.; and Dr. Dennis Morrison, NASA Johnson Space Center, process one of the experiments carried on mission STS-107. Several experiments were found during the search for Columbia debris. Included in the Commercial ITA Biomedical Experiments payload on mission STS-107 are urokinase cancer research, microencapsulation of drugs, the Growth of Bacterial Biofilm on Surfaces during Spaceflight (GOBBSS), and tin crystal formation. The latter was sponsored by the Pembroke Pines Charter Middle School.

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.

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

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

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

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.

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.

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.

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.

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 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.

Novel simultaneous combination chemical thrombolysis/rheolytic thrombectomy therapy for acute critical limb ischemia: the power-pulse spray technique.

PubMed

Allie, David E; Hebert, Chris J; Lirtzman, Mitchell D; Wyatt, Charles H; Keller, V Antoine; Khan, Mohamed H; Barker, Esmond A; McElderry, Michael W; Khan, Muhammad A; Fail, Peter S; Stagg, Samuel J; Mitran, Elena V; Chaisson, Gary; Allie, Sonja D; Allie, Adam A; Walker, Craig M

2004-12-01

The novel power-pulse spray (P-PS) technique maximizes and combines the advantages and minimizes the disadvantages of both chemical thrombolysis (CT) and rheolytic thrombectomy (RT). Forty-nine consecutive patients with iliofemoral thrombotic occlusion were treated via P-PS technique. Using a 6 Fr RT catheter, saline prime was exchanged for thrombolytic solution [group 1, 10-20 mg tenecteplase (TNK)/50 cc saline, n = 25; group 2, 1,000,000 urokinase (UK)/50 cc saline, n = 24]. The outflow port was closed, then the catheter was advanced at 1 mm increments while pulsing lytic agent. After 30-min lysis time, RT and definitive treatment of the underlying stenosis were performed. Procedure success was 23/25 (92%) and 22/24 (91.6%) for group 1 and 2, respectively. The mean total procedure time was 72 and 75 min in group 1 and 2, respectively. Thirty-day limb salvage was 91% in both groups. There were no major surgical complications. The P-PS technique is safe and effective using either UK or TNK, offering several potential advantages over monotherapy, including more rapid revascularization, decreases systemic lytic exposure and bleeding complications while facilitating both CT and RT capacity and efficacy. (c) 2004 Wiley-Liss, Inc.

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 cornea: stasis and dynamics].

PubMed

Nishida, Teruo

2008-03-01

The physiological roles of the cornea are to conduct external light into the eye, focus it, together with the lens, onto the retina, and to provide rigidity to the entire eyeball. Good vision thus requires maintenance of the transparency and proper refractive shape of the cornea. Although the cornea appears to be a relatively static structure, dynamic processes operate within and around the cornea at the tissue, cell, and molecular level. In this article, I review the mechanisms responsible for maintenance of corneal homeostasis as well as the development of new modes of treatment for various corneal diseases. I. The static cornea: structure and physiological functions. The cornea is derived from ectoderm, so that it can be considered as transparent skin. It is devoid of blood vessels and manifests the highest sensitivity in the entire body. The surface of the cornea is covered by tear fluid, which serves both as a lubricant and as a conduit for regulatory molecules. The cornea is also supplied with oxygen and various nutrients by the aqueous humor and a loop vascular system in addition to tear fluid. The cornea interacts with its surrounding tissues directly as well as indirectly through tear fluid or aqueous humor, with such interactions playing an important role in the regulation of corneal structure and functions. The resident cells of the cornea-epithelial cells, fibroblasts (keratocytes), and endothelial cells--also engage in mutual interactions through network systems. These interactions as well as those with infiltrated cells and regulation by nerves contribute to the maintenance of the normal structure and functions of the cornea as well as to the repair of corneal injuries. II. The dynamic cornea: maintenance of structure and functions by network systems. Developments in laser and computer technology have allowed observation of the cells and collagen fibers within the cornea. Furthermore, progress in cell and molecular biology has allowed characterization of dynamic network systems-including cell-cell and cell-extracellular matrix interactions as well as cytokines and neural factors-that contribute to the maintenance of corneal transparency and shape. III. Disruption of network systems: persistent corneal epithelial defects and corneal ulcer. Selection of the appropriate treatment for pathologic lesions of the cornea and the accompanying decrease in visual acuity requires localization of the lesion with regard to the epithelium, stroma, or endothelium of the cornea. In certain instances, however, it is not possible to determine the cause of the problem within the cornea. In such cases, the cause of the pathologic lesion and the target for treatment may lie in the surrounding tissues or environment. For example, corneal epithelial wound healing may be delayed, leading to the development of persistent epithelial defects, as a result of disruption of intercellular junctions between epithelial cells, an abnormality of the corneal basement membrane, altered concentrations of various cytokines in tear fluid, a lowered corneal sensation, or allergic reactions in the lid conjunctiva. Loss of corneal epithelial barrier function can further allow inflammatory cytokines present in tear fluid, together with infiltrated cells, to activate keratocytes and elicit excessive degradation of collagen in the stroma, thereby giving rise to corneal ulcer. IV. Development of new drugs for corneal diseases. We have attempted to apply the results of basic scientific research to the development of new drugs for corneal diseases that remain difficult to treat. The process of authorization for new drugs from the Ministry of Health, Labor, and Welfare takes more than two decades, however. The path from the bench to clinical practice is thus a long one. 1. Development of eyedrops for treatment of persistent corneal epithelial defects. We demonstrated the clinical efficacy of fibronectin eyedrops for the treatment of persistent epithelial defects of the cornea. However, the possibility of blood-borne infections has interfered with the development of serum-derived fibronectin as a drug. An automated machine for the preparation of autologous fibronectin eyedrops has therefore recently been developed. Furthermore, in seeking an alternative to fibronectin eyedrops, we are investigating the effects of a peptide corresponding to the second cell-binding domain of fibronectin on corneal epithelial wound healing. Considering that urokinase-type plasminogen activator may be expressed at the site of corneal epithelial defects and facilitates epithelial migration, the potential clinical application of annexin V, which stimulates the secretion of urokinase-type plasminogen activator for the treatment of persistent corneal epithelial defects is also now under investigation in Japan. 2. Development of eyedrops for treatment of neurotrophic keratopathy. Substance P, a neurotransmitter, stimulates corneal epithelial migration in a synergistic manner with insulin-like growth factor (IGF)--1. We have shown that eyedrops containing both the substance P-derived peptide FGLM-amide and the IGF-1--derived peptide SSSR are effective for the treatment of persistent corneal epithelial defects in individuals with diabetic keratopathy or neurotrophic keratopathy, both of which are associated with a reduction in corneal sensation. 3. Development of drugs for corneal ulcer. Treatment of corneal infection with antibiotics does not necessarily halt the process of corneal ulceration, which is characterized by excessive degradation of stromal collagen, or resolve persistent corneal epithelial defects. In addition to eyedrops for the treatment of persistent corneal epithelial defects, we have therefore also been working on the development of new drugs for the treatment of corneal ulcer. To this end, we have established an experimental system in which corneal fibroblasts are cultured in a three-dimensional collagen gel. With this system, we have shown that triptolide and steroids inhibit collagen degradation by corneal fibroblasts. Triptolide or its derivatives are thus potential drugs for the treatment of corneal ulcer and would work by acting directly on corneal fibroblasts rather than by inhibiting the secreted enzymes(matrix metalloproteinases) responsible for collagen degradation.

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.

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

Morphologic characteristics of central pulmonary thromboemboli predict haemodynamic response in massive pulmonary embolism.

PubMed

Podbregar, Matej; Voga, Gorazd; Krivec, Bojan

2004-08-01

On hospital admission, the morphology of the central pulmonary artery thromboemboli is an independent predictor of 30-day mortality in patients with massive pulmonary embolism (MPE). This may be due to the differential susceptibility of thromboemboli to thrombolysis. The aim of this study was to assess haemodynamic response to treatment in patients with MPE and morphologically different thromboemboli. Prospective observational study. An 11-bed closed medical ICU at a 860-bed community general hospital. Twelve consecutive patients with shock or hypotension due to MPE and central pulmonary thromboemboli detected by transesophageal echocardiography who were treated with thrombolysis between January 2000 through April 2002. Patients were divided into two groups according to the characteristics of detected central pulmonary thromboemboli: group 1, thrombi with one or more long, mobile parts; and group 2, immobile thrombi. Urokinase infusion was terminated when mixed venous oxygen saturation was stabilized above 60% for 15 min. At 2 h, the total pulmonary vascular resistance index was reduced more in group 1 than group 2 [from 27+/-12 mmHg/(l.min.m(2)) to 14+/-6 mmHg/(l.min.m(2)) (-52%) vs 27+/-8 mmHg/(l.min.m(2)) to 23+/-10 mmHg/(l.min.m(2)) (-15%), respectively, P=0.04]. In group 1 thrombolysis was terminated earlier than group 2 (89+/-40 min vs 210+/-62 min, respectively, P= 0.0024). The cumulative dose of urokinase used in group 1 was lower than group 2 (1.7+/-0.3 M i.u. vs 2.7+/-0.5 M i.u., respectively, P= 0.023). Haemodynamic stabilization is achieved faster in patients with mobile central thromboemboli detected by transesophageal echocardiography during MPE.

pH-responsive and enzymatically-responsive hydrogel microparticles for the oral delivery of therapeutic proteins: Effects of protein size, crosslinking density, and hydrogel degradation on protein delivery.

PubMed

Koetting, Michael Clinton; Guido, Joseph Frank; Gupta, Malvika; Zhang, Annie; Peppas, Nicholas A

2016-01-10

Two potential platform technologies for the oral delivery of protein therapeutics were synthesized and tested. pH-responsive poly(itaconic acid-co-N-vinyl-2-pyrrolidone) (P(IA-co-NVP)) hydrogel microparticles were tested in vitro with model proteins salmon calcitonin, urokinase, and rituximab to determine the effects of particle size, protein size, and crosslinking density on oral delivery capability. Particle size showed no significant effect on overall delivery potential but did improve percent release of encapsulated protein over the micro-scale particle size range studied. Protein size was shown to have a significant impact on the delivery capability of the P(IA-co-NVP) hydrogel. We show that when using P(IA-co-NVP) hydrogel microparticles with 3 mol% tetra(ethylene glycol) dimethacrylate crosslinker, a small polypeptide (salmon calcitonin) loads and releases up to 45 μg/mg hydrogel while the mid-sized protein urokinase and large monoclonal antibody rituximab load and release only 19 and 24 μg/mg hydrogel, respectively. We further demonstrate that crosslinking density offers a simple method for tuning hydrogel properties to variously sized proteins. Using 5 mol% TEGDMA crosslinker offers optimal performance for the small peptide, salmon calcitonin, whereas lower crosslinking density of 1 mol% offers optimal performance for the much larger protein rituximab. Finally, an enzymatically-degradable hydrogels of P(MAA-co-NVP) crosslinked with the peptide sequence MMRRRKK were synthesized and tested in simulated gastric and intestinal conditions. These hydrogels offer ideal loading and release behavior, showing no degradative release of encapsulated salmon calcitonin in gastric conditions while yielding rapid and complete release of encapsulated protein within 1h in intestinal conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

A biological tissue adhesive and dissolvent system for intraocular tumor plaque radiotherapy: an in vivo animal model experiment.

PubMed

Zloto, Ofira; Alezra, Dror; Sagiv, Oded; Belkin, Michael; Dai, Vicktoria Vishnevskia; Moroz, Iris; Greenberg, Gahl; Ben-Artsi, Elad; Fabian, Ido Didi

2015-11-01

To examine a novel biological adhesive and dissolvent system for plaque placement and removal using fibrin glue and urokinase, respectively, in an in vivo animal model. The study was performed on 23 rabbit eyes. Of these, eight underwent a technical feasibility study and ultrasonographic plaque displacement measurements, nine were examined clinically and by magnetic resonance imaging and histopathology for tissue reaction to the biological substances used, and in six the impact of fibrin glue as an orbital space occupier on intraocular pressure was assessed. In an additional ex vivo experiment, the glue's radiation attenuating properties were tested using an oncology EDR2 film. Plaque horizontal movement throughout follow-up (7-10 days) was negligible (0.5 ± 0.2 mm), and there was no tilting whatsoever. In the tissue response experiment, no adverse effects were recorded after application of fibrin or urokinase throughout the 21-day follow-up period. Interestingly, a circumscribed local inflammatory response was noted in tissue surrounding the fibrin glue, and persisted at 21 days. In the orbital space-occupying experiment, application of 1 cc fibrin glue did not cause a significant elevation in intraocular pressure (IOP) (P = 0.06), and in the ex vivo experiment, there was no significant difference between radiation readings with and without glue separation of the radioactive sources and film (P = 0.065). The adhesive and dissolvent system was feasible and safe for plaque placement and removal. It may be superior to conventional surgical plaque placement methods in eliminating the relatively common risk of plaque tilting and complications due to scleral suturing.

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.

Microcapsules and Methods for Making

NASA Technical Reports Server (NTRS)

Morrison, Dennis R. (Inventor); Mosier, Benjamin (Inventor)

1994-01-01

This invention relates to methods for forming multi-lamellar microcapsules of both hydrophilic and hydrophobic immiscible liquid phases using several polymer/solvent systems. Liquid-Liquid diffusion and spontaneous emulsification are controlled by properly timed sequence exposures of immiscible phases in aqueous vehicles dispersed in hydrocarbon solvents containing small quantities of oil, co-surfactants, and glycerides. Water-in-oil and oil-in-water microcapsules are formed containing selected combinations of several types of drugs, co-encapsulated within fluid compartments inside the microcapsule. Commercial applications of the process and the resultant product relate to drug therapy for treating medical conditions such as cancer, circulatory conditions, and other conditions in which pharmaceuticals are advantageously targeted to specific organs, or delivered in combination with other pharmaceuticals. Small microcapsules may be delivered intravenously to diseased organs or clotted vessels. The use of multiple drugs within the same microcapsule structure provides advantages for applications such as chemoembolization treatments and may be used to deliver both chemotherapeutic drugs, against tumor cells, and an immuno-adjuvant or immunological stimulant to enhance the patient's immune response. Active forms of urokinase and other enzymes may be delivered without dilution to the local site of an embolism for dissolving the embolism. Thus, the invention has several potentially valuable commercial applications related to pharmaceutical and medical applications.

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.

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

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.

The biochemical and functional food properties of the bowman-birk inhibitor.

PubMed

Losso, Jack N

2008-01-01

The Bowman-Birk inhibitor (BBI) is a small water-soluble protein present in soybean and almost all monocotyledonous and dicotyledonous seeds. The molecular size of BBI ranges from 1,513 Da to about 20,000 Da. BBI is to seeds what alpha(1)-antitrypsin is to humans. Soy-based food products rich in BBI include soybean grits, soymilk, oilcake, soybean isolate, and soybean protein concentrate. BBI is stable within the pH range encountered in most foods, can withstand boiling water temperature for 10 min, resistant to the pH range and proteolytic enzymes of the gastrointestinal tract, bioavailable, and not allergenic. BBI reduces the proteolytic activities of trypsin, chymotrypsin, elastase, cathepsin G, and chymase, serine protease-dependent matrix metalloproteinases, urokinase protein activator, mitogen activated protein kinase, and PI3 kinase, and upregulates connexin 43 (Cx43) expression. Several studies have demonstrated the efficacy of BBI against tumor cells in vitro, animal models, and human phase IIa clinical trials. FDA considers BBI as a drug. In 1999, FDA allowed a health claim on food labels stating that a daily diet containing 25 grams of soy protein, also low in saturated fat and cholesterol, may reduce the risk of heart disease [corrected] This review highlights the biochemical and functional food properties of the Bowman-Birk inhibitor.

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.

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

An anthrax toxin variant with an improved activity in tumor targeting

PubMed Central

Wein, Alexander N.; Peters, Diane E.; Valivullah, Zaheer; Hoover, Benjamin J.; Tatineni, Aparna; Ma, Qian; Fattah, Rasem; Bugge, Thomas H.; Leppla, Stephen H.; Liu, Shihui

2015-01-01

Anthrax lethal toxin (LT) is an A-B type toxin secreted by Bacillus anthracis, consisting of the cellular binding moiety, protective antigen (PA), and the catalytic moiety, lethal factor (LF). To target cells, PA binds to cell-surface receptors and is then proteolytically processed forming a LF-binding competent PA oligomer where each LF binding site is comprised of three subsites on two adjacent PA monomers. We previously generated PA-U2-R200A, a urokinase-activated PA variant with LF-binding subsite II residue Arg200 mutated to Ala, and PA-L1-I210A, a matrix metalloproteinase-activated PA variant with subsite III residue Ile210 mutated to Ala. PA-U2-R200A and PA-L1-I210A displayed reduced cytotoxicity when used singly. However, when combined, they formed LF-binding competent heterogeneous oligomers by intermolecular complementation, and achieved high specificity in tumor targeting. Nevertheless, each of these proteins, in particular PA-L1-I210A, retained residual LF-binding ability. In this work, we screened a library containing all possible amino acid substitutions for LF-binding site to find variants with activity strictly dependent upon intermolecular complementation. PA-I207R was identified as an excellent replacement for the original clockwise-side variant, PA-I210A. Consequently, the new combination of PA-L1-I207R and PA-U2-R200A showed potent anti-tumor activity and low toxicity, exceeding the performance of the original combination, and warranting further investigation. PMID:26584669

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.

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.

Identification of CD147 (basigin) as a mediator of trophoblast functions.

PubMed

Lee, Cheuk-Lun; Lam, Maggie P Y; Lam, Kevin K W; Leung, Carmen O N; Pang, Ronald T K; Chu, Ivan K; Wan, Tiffany H L; Chai, Joyce; Yeung, William S B; Chiu, Philip C N

2013-11-01

Does CD147 regulate trophoblast functions in vitro? CD147 exists as a receptor complex on human trophoblast and regulates the implantation, invasion and differentiation of trophoblast. CD147 is a membrane protein implicated in a variety of physiological and pathological conditions due to its regulation of cell-cell recognition, cell differentiation and tissue remodeling. Reduced placental CD147 expression is associated with pre-eclampsia, but the mechanism of actions remains unclear. A loss of function approach or functional blocking antibody was used to study the function of CD147 in primary human cytotrophoblasts isolated from first trimester termination of pregnancy and/or in the BeWo cell line, which possesses characteristics of human cytotrophoblasts. CD147 expression was analyzed by immunofluorescence staining and western blotting. CD147-associated protein complex on plasma membrane were separated by blue native gel electrophoresis and identified by reversed-phase liquid chromatography coupled with quadrupole time-of-flight hybrid mass spectrometer. Cell proliferation and invasion were determined by fluorometric cell proliferation assays and transwell invasion assays, respectively. Matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA) activities were measured by gelatin gel zymography and uPA assay kits, respectively. Cell migration was determined by wound-healing assays. Cell fusion was analyzed by immunocytochemistry staining of E-cadherin and 4',6-diamidino-2-phenylindole. The transcripts of matrix proteinases and trophoblast lineage markers were measured by quantitative PCR. Extracellular signal-regulated kinase (ERK) activation was analyzed by western blot using antibodies against ERKs. CD147 exists as protein complexes on the plasma membrane of primary human cytotrophoblasts and BeWo cells. Several known CD147-interacting partners, including integrin β1 and monocarboxylate transporter-1, were identified. Suppression of CD147 by siRNA significantly (P < 0.05) reduced trophoblast-endometrial cell interaction, cell invasion, syncytialization, differentiation and ERK activation of BeWo cells. Consistently, anti-CD147 functional blocking antibody suppressed the invasiveness of primary human cytotrophoblasts. The reduced invasiveness was probably due to the restrained (P < 0.05) enzyme activities of MMP-2, MMP-9 and uPA. Most of the above findings are based on BeWo cell lines. These results need to be confirmed with human first trimester primary cytotrophoblast. This is the first study on the role of CD147 in trophoblast function. Further investigation on the function of CD147 and its associated protein complexes will enhance our understanding on human placentation. This work was supported in part by the University of Hong Kong Grant 201011159200. The authors have no competing interests to declare.

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

Percutaneous Transhepatic Portography for the Treatment of Early Portal Vein Thrombosis After Surgery

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

Adani, Gian Luigi, E-mail: adanigl@hotmail.com; Baccarani, Umberto; Risaliti, Andrea

2007-11-15

We treated three cases of early portal vein thrombosis (PVT) by minimally invasive percutaneous transhepatic portography. All patients developed PVT within 30 days of major hepatic surgery (one case each of orthotopic liver transplantation, splenectomy in a previous liver transplant recipient, and right extended hepatectomy with resection and reconstruction of the left branch of the portal vein for tumor infiltration). In all cases minimally invasive percutaneous transhepatic portography was adopted to treat this complication by mechanical fragmentation and pharmacological lysis of the thrombus. A vascular stent was also positioned in the two cases in which the thrombosis was related tomore » a surgical technical problem. Mechanical fragmentation of the thrombus with contemporaneous local urokinase administration resulted in complete removal of the clot and allowed restoration of normal blood flow to the liver after a median follow-up of 37 months. PVT is an uncommon but severe complication after major surgery or liver transplantation. Surgical thrombectomy, with or without reconstruction of the portal vein, and retransplantation are characterized by important surgical morbidity and mortality. Based on our experience, minimally invasive percutaneous transhepatic portography should be considered an option toward successful recanalization of early PVT after major liver surgery including transplantation. Balloon dilatation and placement of a vascular stent could help to decrease the risk of recurrent thrombosis when a defective surgical technique is the reason for the thrombosis.« less

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.

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

The biotechnological potential of fibrinolytic enzymes in the dissolution of endogenous blood thrombi.

PubMed

Kotb, Essam

2014-01-01

Formation of endogenous thrombi in blood vessels is one of the leading causes of death in our modern life. According to data provided by the World Health Organization (WHO) in 2000, heart diseases are responsible for 29% of the total mortality rate in the world. For this, a tremendous amount of research has been done in the area of prevention and treatment of these diseases. The classical therapy of these thrombi relies upon the use of antiplatelets, anticoagulants, or even surgeries. Relatively recently, the fibrinolytic enzymes produced by microorganisms, snakes, earthworms, insects, plants, and other organisms are being successfully used in the treatment of blood clots, especially with regard to the direct dissolving action on fibrin in tandem with less cost and side effects in comparison with the first-generation thrombolytic agents, streptokinase and urokinase. Furthermore, recombinant DNA technology has succeeded in improving and decreasing the undesirable effects of the first generation of enzymes. Recombinant PAs or rt-PAs like alteplase, retelase, saruplase, tenecteplase, lanoteplase, and desmoteplase became available in the drug markets with advantages of less binding loci with PAI-1 to avoid degradation while providing faster and more complete reperfusion in a greater number of patients with less risk of bleeding and intracranial hemorrhage. This review is the first to cover all the natural and recombinant thrombolytic agents used in enzyme therapy. © 2014 American Institute of Chemical Engineers.

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.

Surgical procedure and results of cisternal washing therapy for the prevention of cerebral vasospasm following SAH.

PubMed

Nakagomi, Tadayoshi; Furuya, Kazuhide; Nagashima, Hiroshi; Tanaka, Jun-Ichi; Ishii, Teruyuki; Takanashi, Shigehiko; Shinohara, Takeyuki; Watanabe, Fumihiro; Ogawa, Akiko; Fujii, Norio; Tamura, Akira

2011-01-01

In 1994, we started cisternal washing therapy (CWT) using urokinase combined with head-shaking method in order to prevent cerebral vasospasm. In this paper, we showed the surgical procedure for CWT and reported the effect of this therapy in preventing vasospasm following SAH. A total of 332 consecutive cases with Fisher group 3 SAH since 1988 were analyzed. Of these patients, 118 cases (56 cases before 1994 and 62 cases after 1994) had not CWT, and, 214 cases after 1994 had this therapy. All of these patients had clipping surgery within 3 days following SAH, and had postoperative management both with normovolemia and normal to mild hypertension. In these two groups, the incidence of symptomatic vasospasm (transiently symptomatic vasospasm without infarction), cerebral infarction due to vasospasm on CT, and mortality and morbidity (M&M) due to vasospasm were analyzed. In the group without CWT, the incidences of symptomatic vasospasm, cerebral infarction on CT, and M&M due to vasospasm were 4.2%, 28.8%, and 17.8%, respectively. On the other hand, in the group with CWT, they were 3.7%, 6.5%, and 2.8%, respectively. In the patients with CWT, the incidence of cerebral infarction on CT due to vasospasm and M&M due to vasospasm were significantly (p < 0.05) decreased. CWT was effective in preventing cerebral vasospasm.

Urinary coagulation-fibrinolysis, kallirein-kinin systems and kininase in cases of preclampsia.

PubMed

Mutoh, S; Kobayashi, M; Hirata, J; Itoh, N; Maki, M; Komatsu, Y; Yoshida, A; Sasa, H; Kuroda, K; Kikuchi, Y

1992-01-01

Urinary kallikrein and kallikrein activity significantly decreased in cases of preeclampsia (u-kall./CRE.index 42.39 +/- 9.66 ng/mg, u-kall. act./CRE.index 0.26 +/- 0.06 ng/min/mg), and urinary kininase II and kininase activity significantly increased (u-kininase/CRE.index 10.91 +/- 1.26 x 10(-3) IU/min/mg, u-kininase act./CRE.index 506.37 +/- 178.45 pg/min/mg) when compared with those of normal gravidas from 28 weeks to 42 weeks of gestation (u-kall./CRE.index 189.31 +/- 14.17 ng/mg, u-kall. act./CRE index 1.08 +/- 0.10 ng/min/mg, u-kininase/CRE.index 6.24 +/- 0.31 x 10(-3) IU/min/mg, u-kininase act./CRE.index 15.64 +/- 0.10 pg/min/mg). Urinary FPA, B beta 5-42, alpha 2-PI, and alpha 2PI-plasmin-complex (PIC) significantly increased in preeclampsia (u-FPA/CRE.index 23.59 +/- 8.47 ng/mg, u-B beta/CRE.index 105.26 +/- 29.30 ng/mg, u-alpha 2PI/CRE.index 121.53 +/- 43.57 ng/mg, u-PIC/CRE index 278.39 +/- 60.50 ng/mg) when compared with those of normal control group (u-FPA/CRE.index 0.92 +/- 0.04 ng/mg, u-B beta/CRE.index 12.15 +/- 0.44 ng/mg, u-alpha 2PI/CRE.index 4.18 +/- 0.33 ng/mg, u-PIC/CRE.index 5.98 +/- 1.15 ng/mg). Urinary urokinase markedly increased and urinary D-dimer was detected in severe cases of preeclampsia (u-UK/CRE.index 58.20 +/- 43.69 ng/mg, u-D-dimer 54.76 +/- 9.89 ng/ml) when compared with those of normal control group. These findings suggest that deficiency in urinary kinin excretion may induce hypertension in addition to the changes of urinary coagulation-fibrinolysis system that represents the occurrence of either the endothelial cell injury in the glomerulus or the renal tulbular damage in mild cases of preeclampsia, eventually resulting in the intra-renal vascular coagulation.

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

[Portal vein thrombosis associated with hepatic encephalopathy].

PubMed

Iwatani, Nao; Inatomi, Yuichiro; Yonehara, Toshiro; Fujioka, Shodo; Hashimoto, Yoichiro; Hirano, Teruyuki; Uchino, Makoto

2005-03-01

A 54-year-old man who had been administered chlormadinone acetate 3 months after prostatectomy for prostate cancer, acutely developed disorientation and memory disturbance. Six days later, he experienced high grade fever and epigastralgia. He was suspected to have hepatic encephalopathy, because the Fischer ratio was low although serum ammonia level remained normal. Further examinations including abdominal echography and CT scan disclosed a thrombus extending from the portal vein to the superior mesenteric vein together with abnormal collateral vessels originating from the portal vein. He was successfully treated with warfarin potassium, urokinase and heparin sodium. It was suggested that the patient developed hepatic encephalopathy due to portal-systemic circulation shunting secondary to portal vein thrombosis.

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

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.

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

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

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.

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

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

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.

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.

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

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

Diagnosis and treatment of shock due to massive pulmonary embolism: approach with transesophageal echocardiography and intrapulmonary thrombolysis.

PubMed

Krivec, B; Voga, G; Zuran, I; Skale, R; Pareznik, R; Podbregar, M; Noc, M

1997-11-05

To evaluate the diagnostic value of transesophageal echocardiography (TEE) as an initial diagnostic tool in shocked patients. The second objective was to study therapeutic impact of intrapulmonary thrombolysis in patients with diagnosed massive pulmonary embolism. Prospective observational study. Medical ICU in 800-bed general hospital. Twenty-four consecutive patients with unexplained shock and distended jugular veins. In 18 patients, right ventricular dilatation with global or segmental hypokinesis was documented. In addition, central pulmonary thromboemboli (12 patients), reduced contrast flow in right pulmonary artery (one patient), and right ventricular free wall akinesis (one patient) were found. No additional echocardiographic findings were apparent in four patients. According to pulmonary scintigraphy or autopsy, sensitivity of TEE for diagnosis of massive pulmonary embolism (MPE) in patients with right ventricular dilatation was 92% and specificity was 100%. In patients without right ventricular dilatation, left ventricular dysfunction (four patients) or cardiac tamponade (two patients) was confirmed. Intrapulmonary thrombolysis was evaluated in 11 of 13 patients with MPE. Two patients died prior to attempted thrombolysis. Three patients received streptokinase and eight received urokinase. Twenty-four hours after beginning of treatment, total pulmonary resistance index significantly decreased for 59% and mean pulmonary artery pressure for 31%. Cardiac index increased for 74%. Nine of 11 patients receiving thrombolysis survived to hospital discharge. Bedside TEE is a valuable tool for diagnosis of MPE. It enables immediate intrapulmonary thrombolysis, which seems to be an effective therapeutic alternative in our group of patients with obstructive shock.

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

Application of Ultrasound Energy as a New Drug Delivery System

NASA Astrophysics Data System (ADS)

Tachibana, Katsuro; Tachibana, Shunro

1999-05-01

Ultrasound has been in use for the last three decades as amodality for diagnostic imaging in medicine. Recently, there have beennumerous reports on the application of nonthermal ultrasound energyfor targeting or controlling drug release. This new concept oftherapeutic ultrasound combined with drugs has led to much excitementin various medical fields. Ultrasound energy can enhance the effectsof thrombolytic agents such as urokinase. Therapeutic ultrasoundcatheters are currently being developed for treatment ofcardiovascular diseases. Devices with ultrasound transducers implantedin transdermal drug patches are also being evaluated for possibledelivery of insulin through the skin. Chemical activation of drugs byultrasound energy for treatment of cancers is another new fieldrecently termed “Sonodynamic Therapy”. Various examples of ultrasoundapplication are under investigation which could lead to revolutionarydrug delivery systems in the future.

Secretory expression of a heterologous nattokinase in Lactococcus lactis.

PubMed

Liang, Xiaobo; Zhang, Lixin; Zhong, Jin; Huan, Liandong

2007-05-01

Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. We developed a novel strategy to express a nattokinase from Bacillus subtilis in a live delivery vehicle, Lactococcus lactis. Promoter P( nisZ) and signal peptide SP(Usp) were used for inducible and secretory expression of nattokinase in L. lactis. Western blotting analysis demonstrated that nattokinase was successfully expressed, and about 94% of the enzyme was secreted to the culture. The recombinant nattokinase showed potent fibrinolytic activity, equivalent to 41.7 urokinase units per milliliter culture. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle L. lactis would facilitate the widespread application of nattokinase in the control and prevention of thrombosis diseases.

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.

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

Successful Thrombolysis and Spasmolysis of Acute Leg Ischemia after Accidental Intra-arterial Injection of Dissolved Flunitrazepam Tablets

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

Radeleff, B., E-mail: Boris_radeleff@med.uni-heidelberg.de; Stampfl, U.; Sommer, C.-M.

2011-10-15

A 37-year-old man with known intravenous drug abuse presented in the surgical ambulatory care unit with acute leg ischemia after accidental intra-arterial injection of dissolved flunitrazepam tablets into the right femoral artery. A combination of anticoagulation, vasodilatation, and local selective and superselective thrombolysis with urokinase was performed to salvage the leg. As a result of the severe ischemia-induced pain, the patient had to be monitored over the complete therapy period on the intensive care unit with permanent administration of intravenous fluid and analgetics. We describe the presenting symptoms and the interventional technique, and we discuss the recent literature regarding themore » management of accidental intra-arterial injection of dissolved flunitrazepam tablets.« less

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 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

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.

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

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

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.

The clinical pattern of nephrotic syndrome in children has no effect on the concentration of soluble urokinase receptor (suPAR) in serum and urine.

PubMed

Ochocińska, Agnieszka; Jarmużek, Wioletta; Janas, Roman

2018-04-23

Concentration of soluble urokinase receptor (suPAR) was regarded as viable marker to differentiate the focal segmental glomerulosclerosis (FSGS) from other glomerulopathies and also as predictive parameter for progression of renal disease. The aim of this study was to evaluate serum and urine (s)(u)suPAR concentration in steroid-sensitive and steroid-resistant nephrotic children treated with different (double and triple-drug) regimens. Overall 43 children were evaluated including 14 patients with steroid-resistant nephrotic syndrome (SRNS) aged 9±6 years and 29 with steroid-sensitive nephrotic syndrome (SSNS) aged 9±5 years, as well as control group (n=59). The concentration of suPAR was measured with ELISA kit (R∧D Systems Inc.). There was no difference in serum suPAR level between SRNS (6404, range: 4613-9575 pg/mL) and SSNS (5745, range: 4666-8246 pg/mL) patients, and also in urinary suPAR: SRNS (2877, range: 847- 19121 pg/mL) and SSNS (2854, range: 328-7434 pg/mL), respectively. There was no statistically significant difference in serum biomarker concentrations between patients with severe course of the disease, in combination therapy, with three drugs: CsA + MMF + Pred (5968, range: 4613-9575 pg/mL) in comparison with patients receiving double therapy: CsA + Pred or MMF + Pred (5449, range: 4666-6623 pg/mL, 5905, range: 5102-6730 pg/mL, respectively). SuPAR concentration in the urine of patients treated with Pred + MMF was lower (1493, range: 328-4444 pg/mL) than in patients receiving Pred + CsA (3193, range: 629-7434 pg/mL), as well as lower than in patients with triple combination of drugs (3318, range: 448-5570 pg/mL), however the difference was not statistically significant. Serum and urine concentration of suPAR did not different between different clinical patterns of nephrotic syndrome in children, regardless the immunosuppressive treatment used. © 2018 MEDPRESS.

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.

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.

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.

Red blood cell decreases of microgravity

NASA Technical Reports Server (NTRS)

Johnson, P. C.

1985-01-01

Postflight decreases in red blood cell mass (RBCM) have regularly been recorded after exposure to microgravity. These 5-25 percent decreases do not relate to the mission duration, workload, caloric intake or to the type of spacecraft used. The decrease is accompanied by normal red cell survivals, increased ferritin levels, normal radioactive iron studies, and increases in mean red blood cell volume. Comparable decreases in red blood cell mass are not found after bed rest, a commonly used simulation of the microgravity state. Inhibited bone marrow erythropoiesis has not been proven to date, although reticulocyte numbers in the peripheral circulation are decreased about 50 percent. To date, the cause of the microgravity induced decreases in RBCM is unknown. Increased splenic trapping of circulating red blood cells seem the most logical way to explain the results obtained.

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

Electrochemical properties of 316L stainless steel with culturing L929 fibroblasts

PubMed Central

Hiromoto, Sachiko; Hanawa, Takao

2005-01-01

Potentiodynamic polarization and impedance tests were carried out on 316L stainless steel with culturing murine fibroblast L929 cells to elucidate the corrosion behaviour of 316L steel with L929 cells and to understand the electrochemical interface between 316L steel and cells, respectively. Potential step test was carried out on 316L steel with type I collagen coating and culturing L929 cells to compare the effects of collagen and L929 cells. The open-circuit potential of 316L steel slightly shifted in a negative manner and passive current density increased with cells, indicating a decrease in the protective ability of passive oxide film. The pitting potential decreased with cells, indicating a decrease in the pitting corrosion resistance. In addition, a decrease in diffusivity at the interface was indicated from the decrease in the cathodic current density and the increase in the diffusion resistance parameter in the impedance test. The anodic peak current in the potential step test decreased with cells and collagen. Consequently, the corrosion resistance of 316L steel decreases with L929 cells. In addition, collagen coating would provide an environment for anodic reaction similar to that with culturing cells. PMID:16849246

Gemcitabine treatment induces endoplasmic reticular (ER) stress and subsequently upregulates urokinase plasminogen activator (uPA) to block mitochondrial-dependent apoptosis in Panc-1 cancer stem-like cells (CSCs).

PubMed

Wang, Li; Zhang, Yi; Wang, Weiguo; Zhu, Yunjie; Chen, Yang; Tian, Bole

2017-01-01

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates. The presence of cancer stem-like cells (CSCs) is believed to be among the underlying reasons for the aggressiveness of PDAC, which contributes to chemoresistance and recurrence. However, the mechanisms that induce chemoresistance and inhibit apoptosis remain largely unknown. We used serum-free medium to enrich CSCs from panc-1 human pancreatic cancer cells and performed sphere formation testing, flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and semi-quantitative western blotting to confirm the stemness of panc-1 CSCs. Hallmarks of endoplasmic reticulum (ER) stress, including IRE1, PERK, ATF4, ATF6α, GRP78 and uPA expression, were detected after gemcitabine treatment. Effects of gemcitabine-induced uPA expression on cell invasion, sphere formation, colony formation and gemcitabine sensitivity were detected. Electrophoretic mobility shift assays (EMSAs) and RNA-immunoprecipitation (RIP) were performed to detect interaction between the uPA mRNA 3'-UTR and mutant p53-R273H expressed by panc-1 CSCs. The effects of upregulated uPA by gemcitabine on apoptosis were detected by Annexin V-FITC/PI staining, and the impact of uPA on small molecule CP-31398-restored mutant p53 transcriptional activity was measured by a luciferase reporter assay. Enriched panc-1 CSCs expressing high levels of CD44 and CD133 also produced significantly higher amounts of Oct4 and Nanog. Compared with panc-1 cells, panc-1 CSCs presented chemoresistance to gemcitabine. ER stress gene detections demonstrated effects of gemcitabine-induced ER stress on both the pro-apoptotic and pro-survival branches. ER stress-induced ATF6α upregulated level of uPA by transcriptionally activating GRP78. Gemcitabine-induced uPA promoted invasion, sphere formation and colony formation and attenuated apoptosis induced by gemcitabine in panc-1 CSCs, depending on interaction with mutant p53-R273H. Upregulation of uPA abolished CP-31398-mediated restoration of mutant p53 transcriptional activity in panc-1 CSCs. Gemcitabine treatment induced ER stress and promoted mutant p53-R273H stabilization via transcriptionally activated uPA which may contribute to chemoresistance to gemcitabine. Notably, upregulation of uPA by gemcitabine treatment may lead to the failure of CP-31398; thus, a novel strategy for modulating mutant p53 function needs to be developed.

Gemcitabine treatment induces endoplasmic reticular (ER) stress and subsequently upregulates urokinase plasminogen activator (uPA) to block mitochondrial-dependent apoptosis in Panc-1 cancer stem-like cells (CSCs)

PubMed Central

Wang, Weiguo; Zhu, Yunjie; Chen, Yang

2017-01-01

Background Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates. The presence of cancer stem-like cells (CSCs) is believed to be among the underlying reasons for the aggressiveness of PDAC, which contributes to chemoresistance and recurrence. However, the mechanisms that induce chemoresistance and inhibit apoptosis remain largely unknown. Methods We used serum-free medium to enrich CSCs from panc-1 human pancreatic cancer cells and performed sphere formation testing, flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and semi-quantitative western blotting to confirm the stemness of panc-1 CSCs. Hallmarks of endoplasmic reticulum (ER) stress, including IRE1, PERK, ATF4, ATF6α, GRP78 and uPA expression, were detected after gemcitabine treatment. Effects of gemcitabine-induced uPA expression on cell invasion, sphere formation, colony formation and gemcitabine sensitivity were detected. Electrophoretic mobility shift assays (EMSAs) and RNA-immunoprecipitation (RIP) were performed to detect interaction between the uPA mRNA 3’-UTR and mutant p53-R273H expressed by panc-1 CSCs. The effects of upregulated uPA by gemcitabine on apoptosis were detected by Annexin V-FITC/PI staining, and the impact of uPA on small molecule CP-31398-restored mutant p53 transcriptional activity was measured by a luciferase reporter assay. Results Enriched panc-1 CSCs expressing high levels of CD44 and CD133 also produced significantly higher amounts of Oct4 and Nanog. Compared with panc-1 cells, panc-1 CSCs presented chemoresistance to gemcitabine. ER stress gene detections demonstrated effects of gemcitabine-induced ER stress on both the pro-apoptotic and pro-survival branches. ER stress-induced ATF6α upregulated level of uPA by transcriptionally activating GRP78. Gemcitabine-induced uPA promoted invasion, sphere formation and colony formation and attenuated apoptosis induced by gemcitabine in panc-1 CSCs, depending on interaction with mutant p53-R273H. Upregulation of uPA abolished CP-31398-mediated restoration of mutant p53 transcriptional activity in panc-1 CSCs. Conclusion Gemcitabine treatment induced ER stress and promoted mutant p53-R273H stabilization via transcriptionally activated uPA which may contribute to chemoresistance to gemcitabine. Notably, upregulation of uPA by gemcitabine treatment may lead to the failure of CP-31398; thus, a novel strategy for modulating mutant p53 function needs to be developed. PMID:28854261

Self-reported fatigue common among optimally treated HIV patients: no correlation with cerebral FDG-PET scanning abnormalities.

PubMed

Andersen, Ase B; Law, Ian; Ostrowski, Sisse R; Lebech, Anne Mette; Høyer-Hansen, Gunilla; Højgaard, Liselotte; Gerstoft, Jan; Ullum, Henrik; Kjaer, Andreas

2006-01-01

It was the aim of this study to determine the prevalence and severity of fatigue among optimally treated HIV patients and to investigate the potential association with systemic inflammation and abnormalities of the distribution of cerebral glucose metabolism. A cohort of HIV patients (n = 95), known to be HIV positive for 5 years, on anti-retroviral therapy for a minimum of 3 years and with CD4 counts above 0.2 x 10(9) cells/l, completed a validated fatigue inventory, and plasma was analysed for pro-inflammatory markers including tumour necrosis factor-alpha, interleukin 6 and soluble urokinase receptor (suPAR) levels. The distribution of the regional cerebral metabolic rate of glucose was measured in a sub-group of patients suffering from severe fatigue (n = 9) and a group with no fatigue (n = 7) using fluorine-18-fluorodeoxyglucose positron emission tomography (FDG-PET) scanning. Fifteen percent suffered from severe fatigue, but no association with pro-inflammatory markers was found. About 50% of the FDG-PET-scanned patients showed minor abnormalities in the relative cerebral metabolic rate of glucose. These abnormalities were not associated with fatigue but tended to correlate with a short HIV history (p = 0.058), a low CD4 nadir (p = 0.082) and elevated tumour necrosis factor-alpha levels (p = 0.074). Fatigue is common among optimally treated HIV patients. FDG-PET-described signs of imminent neurodegeneration among HIV patients who had a low CD4 nadir may illustrate an aspect of HIV neuropathogenicity.

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.

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

[MRSA-related empyema as thoracic surgical site infection].

PubMed

Mizutani, Hisao

2009-09-01

The incidence of empyema as a thoracic surgical site infection (SSI) is relating low, but empyema related to MRSA poses an unenviable therapeutic challenge. We review 3 cases of MRSA-related empyema as SSI seem in the last 10 years, and evaluate therapeutic measures. All 3 subjects began being administered vancomycin (VCM) systemically once the diagnosis was established. Subject 1 developed MRSA-related empyema following pulmonary segmentectomy for small-cell lung cancer. The subject was treated following a diagnosis of incisional SSI, with delayed adequate pleural drainage, resulting in treatment difficulties, but was cured without becoming MRSA-negative. Subject 2 developed MRSA-related empyema following pulmonary lobectomy for advanced lung cancer associated with pneumoconiosis. Following bronchoplasty, a chest tube was placed for long-term drainage. The subject did not become MRSA-negative after VCM administration, but became so after linezolid treatment, facilitating a cure. Subject 3, who had secondary pneumothorax, underwent thoracoscopic partial hepatic resection. Intraoperative findings suggested pleural cavity infection, necessitating a prophylactic drain, but MRSA-related pyothorax developed. Fibrinolysis with urokinase effectively cleared up the poor drainage and the subject was cured without becoming MRSA-negative. In conclusion, in controlling MRSA-related empyema as SSI noted that: (1) long-term postperative thoracic drain retention may lead to retrograde infection; (2) surgical procedures reducing the extent of pulmonary resection may effectively prevent pyothorax progression; (3) for poor drainage in advanced pyothorax, fibrinolytic therapy is worth attempting before thoracoscopic surgery; and (4) the timing for discontinuing anti-MRSA drugs should be determined based on the clinical course rather than negative conversion of bacteria.

Destruction of newly released red blood cells in space flight

NASA Technical Reports Server (NTRS)

Alfrey, C. P.; Udden, M. M.; Huntoon, C. L.; Driscoll, T.

1996-01-01

Space flight results in a rapid change in total blood volume, plasma volume, and red blood cell mass because the space to contain blood is decreased. The plasma volume and total blood volume decreases during the first hours in space and remain at a decreased level for the remainder of the flight. During the first several hours following return to earth, plasma volume and total blood volume increase to preflight levels. During the first few days in space recently produced red blood cells disappear from the blood resulting in a decrease in red blood cell mass of 10-15%. Red cells 12 d old or older survive normally and production of new cells continues at near preflight levels. After the first few days in space, the red cell mass is stable at the decreased level. Following return to earth the hemoglobin and red blood cell mass concentrations decrease reflecting the increase in plasma volume. The erythropoietin levels increase responding to "postflight anemia"; red cell production increases, and the red cell mass is restored to preflight levels after several weeks.

Decreased natural killer cell activity in atopic eczema.

PubMed Central

Hall, T J; Rycroft, R; Brostoff, J

1985-01-01

We have studied NK cell activity in atopic and non-atopic subjects using a standard 51Cr-release assay and K562 target cells. In atopics (AT) with allergic rhinitis and/or asthma, NK cell activity was similar to that in non-atopic (N) subjects, whilst patients with severe atopic eczema (AE) had depressed NK cell activity compared to AT or N subjects. In addition, circulating T-cell numbers and Con A responsiveness was decreased in AE, although neither parameter was correlated with decreased NK cell activity. However, decreased NK cell activity in atopic eczema was positively correlated with decreased numbers of Fc gamma + lymphocytes (P = 0.01) and decreased effector: target cell binding (P = 0.05), and negatively correlated with increased monocytes in AE (P = 0.09). AE NK cell activity was equally or more sensitive to the inhibitory effects of drugs such as dibutyryl cyclic AMP, prostaglandins (PG) D2,E2 and histamine. The relative percentage increase in NK cell activity by the interferon inducer poly I:C was similar in AE patients and controls. The results suggest that reduced numbers of circulating NK cells and pre-NK cells account for the depressed level of NK cell activity in subjects with severe atopic eczema. PMID:3876984

Effects of DDT and Triclosan on Tumor-cell Binding Capacity and Cell-Surface Protein Expression of Human Natural Killer Cells

PubMed Central

Hurd-Brown, Tasia; Udoji, Felicia; Martin, Tamara; Whalen, Margaret M.

2012-01-01

1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) and triclosan (TCS) are organochlorine (OC) compounds that contaminate the environment, are found in human blood, and have been shown to decrease the tumor-cell killing (lytic) function of human natural killer (NK) cells. NK cells defend against tumor cells and virally infected cells. They bind to these targets, utilizing a variety of cell surface proteins. This study examined concentrations of DDT and TCS that decrease lytic function for alteration of NK binding to tumor targets. Levels of either compound that caused loss of binding function were then examined for effects on expression of cell-surface proteins needed for binding. NK cells exposed to 2.5 μM DDT for 24 h (which caused a greater than 55% loss of lytic function) showed a decrease in NK binding function of about 22%, and a decrease in CD16 cell-surface protein of 20%. NK cells exposed to 5 μM TCS for 24 h showed a decrease in ability to bind tumor cells of 37% and a decrease in expression of CD56 of about 34%. This same treatment caused a decrease in lytic function of greater than 87%. These results indicated that only a portion of the loss of NK lytic function seen with exposures to these compounds could be accounted for by loss of binding function. They also showed that loss of binding function is accompanied by a loss cell-surface proteins important in binding function. PMID:22729613

Down-regulation of Rab5 decreases characteristics associated with maintenance of cell transformation

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

Silva, Patricio; Soto, Nicolás; Díaz, Jorge

2015-08-21

The early endosomal protein Rab5 is highly expressed in tumor samples, although a causal relationship between Rab5 expression and cell transformation has not been established. Here, we report the functional effects of targeting endogenous Rab5 with specific shRNA sequences in different tumor cell lines. Rab5 down-regulation in B16-F10 cells decreased tumor formation by subcutaneous injection into C57/BL6 mice. Accordingly, Rab5 targeting in B16-F10 and A549, but not MDA-MB-231 cells was followed by decreased cell proliferation, increased apoptosis and decreased anchorage-independent growth. These findings suggest that Rab5 expression is required to maintain characteristics associated with cell transformation. - Highlights: • Rab5more » is important to the maintenance of cell transformation characteristics. • Down-regulation of Rab5 decreases cell proliferation and increases apoptosis in different cancer cells. • Rab5 is required for anchorage-independent growth and tumorigenicity in-vivo.« less

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.

Microbial viability in preparations packaged for single use.

PubMed

Obayashi, Akiko; Oie, Shigeharu; Kamiya, Akira

2003-05-01

We evaluated microbial viability in preparations packaged for single use only which mandate that residual solution be discarded such as albumin and globulin preparations as blood products, preparations containing albumin (such as urokinase and interferon), fat emulsions, and a preparation containing fat emulsions (propofol). In most preparations, Serratia marcescens and Burkholderia cepacia proliferated rapidly at 30 degrees C. However, in globulin preparations containing 1-2.25% glycine to prevent protein degradation (Gamma-Venin P, Venilon-I, Globulin Injection, and Ahlbulin), no growth of S. marcescens and B. cepacia was detected over 24 h at 30 degrees C. For globulin preparations containing 1-2.25% glycine, the injunction to "Discard residual solution after the package has been used" in the package inserts can be revised to "It is possible to use residual solution within 24 h after the package has been used with storage in a cool place."

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.

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

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.

Human trabecular meshwork cell volume decrease by NO-independent soluble guanylate cyclase activators YC-1 and BAY-58-2667 involves the BKCa ion channel.

PubMed

Dismuke, William M; Sharif, Najam A; Ellis, Dorette Z

2009-07-01

There is a correlation between cell volume changes and changes in the rate of aqueous humor outflow; agents that decrease trabecular meshwork (TM) cell volume increase the rate of aqueous humor outflow. This study investigated the effects of the nitric oxide (NO)-independent activators of soluble guanylate cyclase (sGC), YC-1, and BAY-58-2667 on TM cell volume and the signal transduction pathways and ion channel involved. Cell volume was measured with the use of calcein AM fluorescent dye, detected by confocal microscopy. Inhibitors and activators of sGC, 3',5'-cyclic guanosine monophosphate (cGMP), protein kinase G (PKG), and the BK(Ca) channel were used to characterize their involvement in the YC-1- and BAY-58-2667-induced regulation of TM cell volume. cGMP was assayed by an enzyme immunoassay. YC-1 (10 nM-200 microM) and BAY-58-2667 (10 nM-100 microM) each elicited a biphasic effect on TM cell volume. YC-1 (1 microM) increased TM cell volume, but higher concentrations decreased TM cell volume. Similarly, BAY-58-2667 (100 nM) increased TM cell volume, but higher concentrations decreased cell volume. The YC-1-induced cell volume decrease was mimicked by 8-Br-cGMP and abolished by the sGC inhibitor ODQ, the PKG inhibitor (RP)-8-Br-PET-cGMP-S, and the BK(Ca) channel inhibitor IBTX. The BAY-58-2667-induced cell volume decrease was mimicked by 8-Br-cGMP and was abolished by the PKG inhibitor and the BK(Ca) channel inhibitor. Unlike the YC-1 response, ODQ potentiated the BAY-58-2667-induced decreases in cell volume. These data suggest that the NO-independent decrease in TM cell volume is mediated by the sGC/cGMP/PKG pathway and involves K(+) efflux.

Decreased frequencies and impaired functions of the CD31+ subpopulation in Treg cells associated with decreased FoxP3 expression and enhanced Treg cell defects in patients with coronary heart disease.

PubMed

Huang, L; Zheng, Y; Yuan, X; Ma, Y; Xie, G; Wang, W; Chen, H; Shen, L

2017-03-01

Coronary heart disease (CHD) is one of the most common types of organ lesions caused by atherosclerosis, in which CD4 + CD25 + forkhead box protein 3 (FoxP3 + ) regulatory T cells (T reg ) play an atheroprotective role. However, T reg cell numbers are decreased and their functions are impaired in atherosclerosis; the underlying mechanisms remain unclear. CD31 plays an important part in T cell response and contributes to maintaining T cell tolerance. The immunomodulatory effects of CD31 are also implicated in atherosclerosis. In this study, we found that decreased frequencies of the CD31 + subpopulation in T reg cells (CD31 + Tr cells) correlated positively with decreased FoxP3 expression in CHD patients. Cell culture in vitro demonstrated CD31 + Tr cells maintaining stable FoxP3 expression after activation and exhibited enhanced proliferation and immunosuppression compared with the CD31 - subpopulation in T reg cells (CD31 - Tr cells). We also confirmed impaired secretion of transforming growth factor (TGF)-β1 and interleukin (IL)-10 in CD31 + Tr cells of CHD patients. Further analysis revealed reduced phospho-SHP2 (associated with CD31 activation) and phospho-signal transducer and activator of transcription-5 (STAT-5) (associated with FoxP3 transcription) levels in CD31 + Tr cells of CHD patients, suggesting that decreased FoxP3 expression in CD31 + Tr cells might be because of attenuated SHP2 and STAT-5 activation. These data indicate that decreased frequencies and impaired functions of the CD31 + Tr subpopulation associated with decreased FoxP3 expression give rise, at least in part, to T reg cell defects in CHD patients. Our findings emphasize the important role of the CD31 + Tr subpopulation in maintaining T reg cell normal function and may provide a novel explanation for impaired immunoregulation of T reg cells in CHD. © 2016 British Society for Immunology.

Butyltin exposure causes a rapid decrease in cyclic AMP levels in human lymphocytes.

PubMed

Whalen, M M; Loganathan, B G

2001-03-15

Natural killer (NK) cells are a subset of lymphocytes that are capable of killing tumor cells, virally infected cells, and antibody-coated cells. Butyltins (BTs) are used in a variety of consumer products and industrial applications. Tributyltin (TBT) is found in dairy products, meat, and fish. Dibutyltin (DBT) is found in plastic products, beverages stored in PVC pipes during manufacturing, and poultry products. BTs appear to increase the risk of cancer and viral infections in exposed individuals. This increased risk may be due in part to the inhibitory effect of these compounds on the cytotoxic function of NK cells. A 24-h exposure of NK cells to 200 nM TBT or 1.5 microM DBT decreased the cytotoxic function of NK cells by greater than 90%. Higher concentrations of TBT and DBT decreased the cytotoxic function of NK cells (by greater than 90%) after only a 1-h exposure. A 24-h exposure to either TBT or DBT decreased intracellular ATP levels by about 30%. However, as much as a 1-h exposure to either 300 nM TBT or 10 microM DBT caused no significant decrease in ATP levels. Thus, a decrease in ATP levels is a longer-term consequence of BT exposure. Intracellular levels of cAMP are decreased by as much as 80% within 5 min of exposure to either TBT or DBT. This rapid decline in cAMP levels in NK cells may be a consequence of BT exposure that is related to the rapid decrease in the cytotoxic function of NK cells. Copyright 2001 Academic Press.

Monocyte-macrophage membrane possesses free radicals scavenging activity: stimulation by polyphenols or by paraoxonase 1 (PON1).

PubMed

Rosenblat, M; Elias, A; Volkova, N; Aviram, M

2013-04-01

In the current study, we analysed free radicals scavenging activity of monocytes-macrophages in the absence or presence of antioxidants such as polyphenols or paraoxonase 1 (PON1). THP-1 human monocytic cell line, murine J774A.1 macrophages, as well as human primary monocytes have the capability to scavenge free radicals, as measured by the 1-diphenyl-2-picryl-hydrazyl (DPPH) assay. This effect (which could be attributed to the cell's membrane) was cell number and incubation time dependent. Upon incubation of J774A.1 macrophages with acetylated LDL (Ac-LDL), with VLDL, or with the radical generator, AAPH, the cells' lipid peroxides content, and paraoxonase 2 (PON2) activity were significantly increased. While non-treated cells decreased DPPH absorbance by 65%, the Ac-LDL-, VLDL- or AAPH-treated cells, decreased it by only 33%, 30%, or 45%, respectively. We next analysed the effect of J774A.1 macrophage enrichment with antioxidants, such as polyphenols or PON1 on the cells' free radicals scavenging activity. Non-treated cells decreased DPPH absorbance by 50%, whereas vitamin E-, punicalagin- or PJ-treated cells significantly further decreased it, by 75%. Similarly, in PON1-treated cells DPPH absorbance was further decreased by 63%, in association with 23% increment in PON1 catalytic activity. In cells under oxidative stress [treated with AAPH-, or with oxidized LDL], PON1 activity was decreased by 31% or 40%, as compared to the activity observed in PON1 incubated with non-treated cells. We conclude that monocytes-macrophages possess free radicals scavenging activity, which is decreased under atherogenic conditions, and increased upon cell enrichment with potent antioxidants such as nutritional polyphenols, or PON1.

IMPACT OF SMOKING HABITS ON THE STATE OF CHROMATIN AND MORPHOLOGY OF BUCCAL EPITHELIAL CELLS AMONG MEDICAL STUDENTS.

PubMed

Volkova, O; Ryabokon, E; Magda, I; Shckorbatov, Y

2017-01-01

The cells of buccal epithelium were investigated in groups of smoking and non-smoking students. Cell samples were collected by scraping with blunt sterile spatula, stained with orcein and photographed. The smoking of cigarettes and hookah induces significant decrease in nuclear and cell perimeter and cell area in cells of buccal epithelium. Smoking of hookah induces, besides, the heterochromatization in cell nuclei and the decrease of nuclear area. The data obtained indicate stress reaction in cells (heterochromatinization) and apoptosis-related changes in cells (decrease of nuclear and cell perimeter and cell area). These data show unfavorable effects of smoking cigarettes and even more harmful effect of hookah smoking.

Targeting of antibody-conjugated plasminogen activators to the pulmonary vasculature.

PubMed

Muzykantov, V R; Barnathan, E S; Atochina, E N; Kuo, A; Danilov, S M; Fisher, A B

1996-11-01

Thrombolytic therapy has not been widely used for pulmonary embolism due to less than optimal results with conventional plasminogen activators. We propose a new approach to deliver plasminogen activators to the luminal surface of the pulmonary vasculature to potentially improve dissolution of pulmonary thromboemboli. Our previous studies have documented that a monoclonal antibody (mAb) to angiotensin-converting enzyme (anti-angiotensin-converting enzyme mAb 9B9) accumulates in the lungs of various animal species after systemic administration. We coupled 125I-labeled biotinylated plasminogen activators (single-chain urokinase plasminogen activator, tissue-type plasminogen activator and streptokinase) to biotinylated mAb 9B9, using streptavidin as a cross-linker. The fibrinolytic activity of plasminogen activators was not changed significantly by either biotinylation or by coupling to streptavidin. Antibody-conjugated plasminogen activators bind to the antigen immobilized in plastic wells and provide lysis of fibrin clots formed in these wells. Therefore, antibody-conjugated plasminogen activators bound to their target antigen retain their capacity to activate plasminogen. One hour after i.v. injection of mAb 9B9-conjugated radiolabeled biotinylated single-chain urokinase plasminogen activator, biotinylated tissue-type plasminogen activator or biotinylated-streptokinase in rats, the level of radiolabel was 7.4 +/- 0.8, 5.9 +/- 0.4 and 3.6 +/- 0.4% of injected dose/g (ID/g) of lung tissue vs. 0.5 +/- 0.01, 0.3 +/- 0.01 and 0.6 +/- 0.3% ID/g after injection of the same activators conjugated with control mouse IgG (P < .01 in all cases). Injection of mAb 9B9-conjugated radiolabeled plasminogen activator led to its rapid pulmonary uptake with a peak value 6.2 +/- 1.2% ID/g attained 3 hr after injection. One day later, 2.2 +/- 0.5% of the injected radioactivity was found per gram of lung tissue, although the blood level was 0.13 +/- 0.03% ID/g (lung/blood ratio 16.7 +/- 0.3). Therefore, conjugation of plasminogen activators with anti-angiotensin-converting enzyme mAb 9B9 provides their specific targeting to and prolonged association with the pulmonary vasculature. These results provide a basis for study of the local pulmonary fibrinolysis by mAb 9B9-conjugated plasminogen activators.

Reducing FLI1 Levels in the MRL/lpr Lupus Mouse Model Impacts T Cell Function by Modulating Glycosphingolipid Metabolism

PubMed Central

Richard, Erin Morris; Thiyagarajan, Thirumagal; Bunni, Marlene A.; Basher, Fahmin; Roddy, Patrick O.; Siskind, Leah J.; Nietert, Paul J.; Nowling, Tamara K.

2013-01-01

Systemic Lupus erythematosus (SLE) is an autoimmune disease caused, in part, by abnormalities in cells of the immune system including B and T cells. Genetically reducing globally the expression of the ETS transcription factor FLI1 by 50% in two lupus mouse models significantly improves disease measures and survival through an unknown mechanism. In this study we analyze the effects of reducing FLI1 in the MRL/lpr lupus prone model on T cell function. We demonstrate that adoptive transfer of MRL/lpr Fli1 +/+ or Fli1 +/- T cells and B cells into Rag1-deficient mice results in significantly decreased serum immunoglobulin levels in animals receiving Fli1 +/- lupus T cells compared to animals receiving Fli1 +/+ lupus T cells regardless of the genotype of co-transferred lupus B cells. Ex vivo analyses of MRL/lpr T cells demonstrated that Fli1 +/- T cells produce significantly less IL-4 during early and late disease and exhibited significantly decreased TCR-specific activation during early disease compared to Fli1 +/+ T cells. Moreover, the Fli1 +/- T cells expressed significantly less neuraminidase 1 (Neu1) message and decreased NEU activity during early disease and significantly decreased levels of glycosphingolipids during late disease compared to Fli1 +/+ T cells. FLI1 dose-dependently activated the Neu1 promoter in mouse and human T cell lines. Together, our results suggest reducing FLI1 in lupus decreases the pathogenicity of T cells by decreasing TCR-specific activation and IL-4 production in part through the modulation of glycosphingolipid metabolism. Reducing the expression of FLI1 or targeting the glycosphingolipid metabolic pathway in lupus may serve as a therapeutic approach to treating lupus. PMID:24040398

Reducing FLI1 levels in the MRL/lpr lupus mouse model impacts T cell function by modulating glycosphingolipid metabolism.

PubMed

Richard, Erin Morris; Thiyagarajan, Thirumagal; Bunni, Marlene A; Basher, Fahmin; Roddy, Patrick O; Siskind, Leah J; Nietert, Paul J; Nowling, Tamara K

2013-01-01

Systemic Lupus erythematosus (SLE) is an autoimmune disease caused, in part, by abnormalities in cells of the immune system including B and T cells. Genetically reducing globally the expression of the ETS transcription factor FLI1 by 50% in two lupus mouse models significantly improves disease measures and survival through an unknown mechanism. In this study we analyze the effects of reducing FLI1 in the MRL/lpr lupus prone model on T cell function. We demonstrate that adoptive transfer of MRL/lpr Fli1(+/+) or Fli1(+/-) T cells and B cells into Rag1-deficient mice results in significantly decreased serum immunoglobulin levels in animals receiving Fli1(+/-) lupus T cells compared to animals receiving Fli1(+/+) lupus T cells regardless of the genotype of co-transferred lupus B cells. Ex vivo analyses of MRL/lpr T cells demonstrated that Fli1(+/-) T cells produce significantly less IL-4 during early and late disease and exhibited significantly decreased TCR-specific activation during early disease compared to Fli1(+/+) T cells. Moreover, the Fli1(+/-) T cells expressed significantly less neuraminidase 1 (Neu1) message and decreased NEU activity during early disease and significantly decreased levels of glycosphingolipids during late disease compared to Fli1(+/+) T cells. FLI1 dose-dependently activated the Neu1 promoter in mouse and human T cell lines. Together, our results suggest reducing FLI1 in lupus decreases the pathogenicity of T cells by decreasing TCR-specific activation and IL-4 production in part through the modulation of glycosphingolipid metabolism. Reducing the expression of FLI1 or targeting the glycosphingolipid metabolic pathway in lupus may serve as a therapeutic approach to treating lupus.

A comparative study of U937 cell size changes during apoptosis initiation by flow cytometry, light scattering, water assay and electronic sizing.

PubMed

Yurinskaya, Valentina; Aksenov, Nikolay; Moshkov, Alexey; Model, Michael; Goryachaya, Tatyana; Vereninov, Alexey

2017-10-01

A decrease in flow cytometric forward light scatter (FSC) is commonly interpreted as a sign of apoptotic cell volume decrease (AVD). However, the intensity of light scattering depends not only on the cell size but also on its other characteristics, such as hydration, which may affect the scattering in the opposite way. That makes estimation of AVD by FSC problematic. Here, we aimed to clarify the relationship between light scattering, cell hydration (assayed by buoyant density) and cell size by the Coulter technique. We used human lymphoid cells U937 exposed to staurosporine, etoposide or hypertonic stress as an apoptotic model. An initial increase in FSC was found to occur in apoptotic cells treated with staurosporine and hypertonic solutions; it is accompanied by cell dehydration and is absent in apoptosis caused by etoposide that is consistent with the lack of dehydration in this case. Thus, the effect of dehydration on the scattering signal outweighs the effect of reduction in cell size. The subsequent FSC decrease, which occurred in parallel to accumulation of annexin-positive cells, was similar in apoptosis caused by all three types of inducers. We conclude that an increase, but not a decrease in light scattering, indicates the initial cell volume decrease associated with apoptotic cell dehydration.

Effectiveness of octreotide in controlling fasting hypergastrinemia and related enterochromaffin-like cell growth.

PubMed

Ferraro, G; Annibale, B; Marignani, M; Azzoni, C; D'Adda, T; D'Ambra, G; Bordi, C; delle Fave, G

1996-02-01

The effects of long term (6-month), high (500-micrograms), once a day administration of octreotide on enterochromaffin-like (ECL) cell proliferation were evaluated in eight patients with hypergastrinemic atrophic gastritis at risk for the development of gastric carcinoids. Fasting gastrin levels were determined during treatment and up to 6 months after the end of treatment. Chromogranin A, hCG alpha, and somatostatin-immunostained cells were morphometrically evaluated in biopsy specimens of corpus mucosa taken before and after treatment. The results showed that gastrin levels significantly decreased from 950 to 238 ng/L (-74.9%; P < 0.01) at the end of treatment, a decrease that persisted 6 months after the end of treatment (450 ng/L; P < 0.05). The volume density of CgA cells (mostly ECL cells) decreased from 3.7% to 2.1% of the epithelial component (-43%; P < 0.014), that of hCG alpha-storing ECL cells decreased by 85% (P < 0.0007), and that of somatostatin-stained cells decreased by 74% (P < 0.04). No clinically significant side-effects were found. It is concluded that octreotide treatment as used in the present study is safe and effective in reducing hypergastrinemia and associated ECL cell changes in patients with atrophic gastritis. The decrease in D cells is consistent with the occurrence of somatostatin receptors and related autocrine regulation in these cells.

Menadione induces G2/M arrest in gastric cancer cells by down-regulation of CDC25C and proteasome mediated degradation of CDK1 and cyclin B1

PubMed Central

Lee, Min Ho; Cho, Yoonjung; Kim, Do Hyun; Woo, Hyun Jun; Yang, Ji Yeong; Kwon, Hye Jin; Yeon, Min Ji; Park, Min; Kim, Sa-Hyun; Moon, Cheol; Tharmalingam, Nagendran; Kim, Tae Ue; Kim, Jong-Bae

2016-01-01

Menadione (vitamin K3) has been reported to induce apoptotic cell death and growth inhibition in various types of cancer cells. However, involvement of menadione in cell cycle control has not been considered in gastric cancer cells yet. In the current study, we have investigated whether menadione is involved in the cell cycle regulation and suppression of growth in gastric cancer cells. In the cell cycle analysis, we found that menadione induced G2/M cell cycle arrest in AGS cells. To elucidate the underlying mechanism, we investigated the cell cycle regulatory molecules involved in the G2/M cell cycle transition. After 24 h of menadione treatment, the protein level of CDK1, CDC25C and cyclin B1 in AGS cells was decreased in a menadione dose-dependent manner. In the time course experiment, the protein level of CDC25C decreased in 6 h, and CDK1and cyclin B1 protein levels began to decrease after 18 h of menadione treatment. We found that mRNA level of CDC25C decreased by menadione treatment in 6 h. Menadione did not have an influence on mRNA level of CDK1 and cyclin B1 though the protein levels were decreased. However, the decreased protein levels of CDK1 and cyclin B1 were recovered by inhibition of proteasome. Collectively, these results suggest that menadione inhibits growth of gastric cancer cells by reducing expression of CDC25C and promoting proteasome mediated degradation of CDK1 and cyclin B1 thereby blocking transition of the cell cycle from G2 phase to M phase. PMID:28077999

Silibinin inhibits triple negative breast cancer cell motility by suppressing TGF-β2 expression.

PubMed

Kim, Sangmin; Han, Jeonghun; Jeon, Myeongjin; You, Daeun; Lee, Jeongmin; Kim, Hee Jung; Bae, Sarang; Nam, Seok Jin; Lee, Jeong Eon

2016-08-01

Transforming growth factor-beta (TGF-β) is a multifunctional cytokine that regulates many biological events including cell motility and angiogenesis. Here, we investigated the role of elevated TGF-β2 level in triple negative breast cancer (TNBC) cells and the inhibitory effect of silibinin on TGF-β2 action in TNBC cells. Breast cancer patients with high TGF-β2 expression have a poor prognosis. The levels of TGF-β2 expression increased significantly in TNBC cells compared with those in non-TNBC cells. In addition, cell motility-related genes such as fibronectin (FN) and matrix metalloproteinase-2 (MMP-2) expression also increased in TNBC cells. Basal FN, MMP-2, and MMP-9 expression levels decreased in response to LY2109761, a dual TGF-β receptor I/II inhibitor, in TNBC cells. TNBC cell migration also decreased in response to LY2109761. Furthermore, we observed that TGF-β2 augmented the FN, MMP-2, and MMP-9 expression levels in a time- and dose-dependent manner. In contrast, TGF-β2-induced FN, MMP-2, and MMP-9 expression levels decreased significantly in response to LY2109761. Interestingly, we found that silibinin decreased TGF-β2 mRNA expression level but not that of TGF-β1 in TNBC cells. Cell migration as well as basal FN and MMP-2 expression levels decreased in response to silibinin. Furthermore, silibinin significantly decreased TGF-β2-induced FN, MMP-2, and MMP-9 expression levels and suppressed the lung metastasis of TNBC cells. Taken together, these results suggest that silibinin suppresses metastatic potential of TNBC cells by inhibiting TGF-β2 expression in TNBC cells. Thus, silibinin may be a promising therapeutic drug to treat TNBC.

Daphnetin inhibits invasion and migration of LM8 murine osteosarcoma cells by decreasing RhoA and Cdc42 expression

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

Fukuda, Hiroki; Nakamura, Seikou; Chisaki, Yugo

2016-02-26

Daphnetin, 7,8-dihydroxycoumarin, present in main constituents of Daphne odora var. marginatai, has multiple pharmacological activities including anti-proliferative effects in cancer cells. In this study, using a Transwell system, we showed that daphnetin inhibited invasion and migration of highly metastatic murine osteosarcoma LM8 cells in a dose-dependent manner. Following treatment by daphnetin, cells that penetrated the Transwell membrane were rounder than non-treated cells. Immunofluorescence analysis revealed that daphnetin decreased the numbers of intracellular stress fibers and filopodia. Moreover, daphnetin treatment dramatically decreased the expression levels of RhoA and Cdc42. In summary, the dihydroxycoumarin derivative daphnetin inhibits the invasion and migration ofmore » LM8 cells, and therefore represents a promising agent for use against metastatic cancer. - Highlights: • Daphnetin, a coumarin-derivative, inhibited invasion and migration of LM8 cells. • Stress fibers and filopodia were decreased by daphnetin treatment. • Daphnetin decreased RhoA and Cdc42 protein expression.« less

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.

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.

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.

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.

Benzyl isothiocyanate alters the gene expression with cell cycle regulation and cell death in human brain glioblastoma GBM 8401 cells.

PubMed

Tang, Nou-Ying; Chueh, Fu-Shin; Yu, Chien-Chih; Liao, Ching-Lung; Lin, Jen-Jyh; Hsia, Te-Chun; Wu, King-Chuen; Liu, Hsin-Chung; Lu, Kung-Wen; Chung, Jing-Gung

2016-04-01

Glioblastoma multiforme (GBM) is a highly malignant devastating brain tumor in adults. Benzyl isothiocyanate (BITC) is one of the isothiocyanates that have been shown to induce human cancer cell apoptosis and cell cycle arrest. Herein, the effect of BITC on cell viability and apoptotic cell death and the genetic levels of human brain glioblastoma GBM 8401 cells in vitro were investigated. We found that BITC induced cell morphological changes, decreased cell viability and the induction of cell apoptosis in GBM 8401 cells was time-dependent. cDNA microarray was used to examine the effects of BITC on GBM 8401 cells and we found that numerous genes associated with cell death and cell cycle regulation in GBM 8401 cells were altered after BITC treatment. The results show that expression of 317 genes was upregulated, and two genes were associated with DNA damage, the DNA-damage-inducible transcript 3 (DDIT3) was increased 3.66-fold and the growth arrest and DNA-damage-inducible α (GADD45A) was increased 2.34-fold. We also found that expression of 182 genes was downregulated and two genes were associated with receptor for cell responses to stimuli, the EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1) was inhibited 2.01-fold and the TNF receptor-associated protein 1 (TRAP1) was inhibited 2.08-fold. BITC inhibited seven mitochondria ribosomal genes, the mitochondrial ribosomal protein; tumor protein D52 (MRPS28) was inhibited 2.06-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein L23 (MRPL23) decreased 2.08-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein S12 (MRPS12) decreased 2.08-fold, the mitochondria ribosomal protein L12 (MRPL12) decreased 2.25-fold and the mitochondria ribosomal protein S34 (MRPS34) was decreased 2.30-fold in GBM 8401 cells. These changes of gene expression can provide the effects of BITC on the genetic level and are potential biomarkers for glioblastoma therapy.

Arecoline decreases interleukin-6 production and induces apoptosis and cell cycle arrest in human basal cell carcinoma cells

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

Huang, Li-Wen; Hsieh, Bau-Shan; Cheng, Hsiao-Ling

2012-01-15

Arecoline, the most abundant areca alkaloid, has been reported to decrease interleukin-6 (IL-6) levels in epithelial cancer cells. Since IL-6 overexpression contributes to the tumorigenic potency of basal cell carcinoma (BCC), this study was designed to investigate whether arecoline altered IL-6 expression and its downstream regulation of apoptosis and the cell cycle in cultured BCC-1/KMC cells. BCC-1/KMC cells and a human keratinocyte cell line, HaCaT, were treated with arecoline at concentrations ranging from 10 to 100 μg/ml, then IL-6 production and expression of apoptosis- and cell cycle progress-related factors were examined. After 24 h exposure, arecoline inhibited BCC-1/KMC cell growthmore » and decreased IL-6 production in terms of mRNA expression and protein secretion, but had no effect on HaCaT cells. Analysis of DNA fragmentation and chromatin condensation showed that arecoline induced apoptosis of BCC-1/KMC cells in a dose-dependent manner, activated caspase-3, and decreased expression of the anti-apoptotic protein Bcl-2. In addition, arecoline induced progressive and sustained accumulation of BCC-1/KMC cells in G2/M phase as a result of reducing checkpoint Cdc2 activity by decreasing Cdc25C phosphatase levels and increasing p53 levels. Furthermore, subcutaneous injection of arecoline led to decreased BCC-1/KMC tumor growth in BALB/c mice by inducing apoptosis. This study demonstrates that arecoline has potential for preventing BCC tumorigenesis by reducing levels of the tumor cell survival factor IL-6, increasing levels of the tumor suppressor factor p53, and eliciting cell cycle arrest, followed by apoptosis. Highlights: ► Arecoline has potential to prevent against basal cell carcinoma tumorigenesis. ► It has more effectiveness on BCC as compared with a human keratinocyte cell line. ► Mechanisms involved including reducing tumor cells’ survival factor IL-6, ► Decreasing Cdc25C phosphatase, enhancing tumor suppressor factor p53, ► Eliciting G2/M phase arrest, followed by apoptosis.« less

Methamphetamine decreases dentate gyrus stem cell self-renewal and shifts the differentiation towards neuronal fate.

PubMed

Baptista, Sofia; Lasgi, Charlène; Benstaali, Caroline; Milhazes, Nuno; Borges, Fernanda; Fontes-Ribeiro, Carlos; Agasse, Fabienne; Silva, Ana Paula

2014-09-01

Methamphetamine (METH) is a highly addictive psychostimulant drug of abuse that negatively interferes with neurogenesis. In fact, we have previously shown that METH triggers stem/progenitor cell death and decreases neuronal differentiation in the dentate gyrus (DG). Still, little is known regarding its effect on DG stem cell properties. Herein, we investigate the impact of METH on mice DG stem/progenitor cell self-renewal functions. METH (10nM) decreased DG stem cell self-renewal, while 1nM delayed cell cycle in the G0/G1-to-S phase transition and increased the number of quiescent cells (G0 phase), which correlated with a decrease in cyclin E, pEGFR and pERK1/2 protein levels. Importantly, both drug concentrations (1 or 10nM) did not induce cell death. In accordance with the impairment of self-renewal capacity, METH (10nM) decreased Sox2(+)/Sox2(+) while increased Sox2(-)/Sox2(-) pairs of daughter cells. This effect relied on N-methyl-d-aspartate (NMDA) signaling, which was prevented by the NMDA receptor antagonist, MK-801 (10μM). Moreover, METH (10nM) increased doublecortin (DCX) protein levels consistent with neuronal differentiation. In conclusion, METH alters DG stem cell properties by delaying cell cycle and decreasing self-renewal capacities, mechanisms that may contribute to DG neurogenesis impairment followed by cognitive deficits verified in METH consumers. Copyright © 2014. Published by Elsevier B.V.

Laser and Non-Coherent Light Effect on Peripheral Blood Normal and Acute Lymphoblastic Leukemic Cells by Using Different Types of Photosensitizers

NASA Astrophysics Data System (ADS)

El Batanouny, Mohamed H.; Khorshid, Amira M.; Arsanyos, Sonya F.; Shaheen, Hesham M.; Abdel Wahab, Nahed; Amin, Sherif N.; El Rouby, Mahmoud N.; Morsy, Mona I.

2010-04-01

Photodynamic therapy (PDT) is a novel treatment modality of cancer and non-cancerous conditions that are generally characterized by an overgrowth of unwanted or abnormal cells. Irradiation of photosensitizer loaded cells or tissues leads via the photochemical reactions of excited photosensitizer molecules to the production of singlet oxygen and free radicals, which initiate cell death. Many types of compounds have been tested as photosensitizers, such as methylene blue (MB) and photopherin seemed to be very promising. This study involved 26 cases of acute lymphoblastic leukemia and 15 normal volunteers as a control group. The cell viability was measured by Light microscope and flowcytometer. Mode of cell death was detected by flowcytometer and electron microscope in selected cases. The viability percentage of normal peripheral blood mononuclear cells (PBMC) incubated with methylene blue (MB) alone or combined with photo irradiation with diode laser (as measured by light microscope) was significantly lower than that of untreated cases either measured after 1 hour (p<0.001) or 24 hours (p<0.001) post incubation. There was a significantly lower viability percentage of normal cells incubated with MB and photoirradiated with diode laser compared to normal cells treated with MB alone for either measured after 1 hour (p<0.001) or 24 hours (p<0.001) post incubation. The decrease in viability was more enhanced with increasing the incubation time. For normal cells incubated with photopherin either for 1/2 an hour or 1 hour, there was a weak cytotoxic effect compared to the effect on untreated cells. There was a significant decrease in viability percentage of cells incubated with photopherin either for 1/2 an hour or 1 hour and photoirradiated with He:Ne laser compared to normal untreated cells. The decrease in the cell viability percentage was significantly lower with the use of PDT (photopherin and He:Ne laser ) compared to either photopherin alone or He:Ne laser alone. The decrease in viability was more enhanced with increasing the incubation time. The same effects reported on normal cells were detected on leukemic cells on comparing different methods used. However a more pronounced decrease in cell viability was detected. The most efficient ways of decreasing viability of leukemic cells with much less effect on normal cells was the use of PDT of cell incubation with MB for 1 hour then photoirradiation with diode laser and PDT of cell incubation with photopherin for 1 hour then photoirradiation with He:Ne laser. Flowcytometer (FCM) was more sensitivite than the light microscope in detecting the decrease in cell viability, it also helped in determining the mode of cell death weather apoptosis, necrosis or combined apoptosis and necrosis. Apoptotic cell percentage was higher in PDT of MB and Diode laser or photopherin and He:Ne laser, treated ALL cells compared to untreated ALL cells after 1 hour but was significantly lower after 24 hours post irradiation. A significant increase in necrotic, combined necrotic and apoptotic cell percentages either measured 1 hour or 24 hours post PDT, compared to untreated ALL cells and PDT treated normal cells. Electron microscope helped in detecting early cellular apoptotic changes occurring in response to different therapeutic modalities used in this study. In conclusion, PDT proved to be an effective clinical modality in decreasing the number of leukemic cells when irradiated in vitro with appropriate laser and photosensitizer system. Both PDT systems used in this study were efficient in inducing cell death of leukemic cells compared to untreated leukemic cells. However, photopherin PDT system was more efficient in decreasing the cell viability. A significant decrease in viability percentage was detected when studying the effect of PDT on leukemic cells compared to that on normal cells. This suggests that PDT when applied clinically will selectively differentiate between leukemic cells and normal cells, offering a successful component in ALL therapy.

Tuberin-deficiency downregulates N-cadherin and upregulates vimentin in kidney tumor of TSC patients

PubMed Central

Liang, Sitai; Salas, Tiffanie; Gencaslan, Emre; Li, Baojie; Habib, Samy L.

2014-01-01

Angiomyolipomas (AMLs) are associated with cell fibrosis in kidney of Tuberous Sclerosis Complex patients. The mechanism by which the fibrotic proteins accumulated in AMLs has not been explored. In the present study, we investigated the role of Akt/tuberin/mTOR pathway in the regulation cell fibrosis proteins. AML cells that expressed low levels of tuberin showed less expression of N-cadherin and higher of vimentin proteins compared to HEK293 cells. AML cells infected with Ad-tuberin showed a significant decrease in vimentin and an increase in N-cadherin protein expression. In addition, cells treated with rapamycin showed a significant increase in p-Akt and a decrease in p-p70S6K that was associated with a decrease expression of vimentin and a slight increase expression in N-cadherin. On the other hand, cells treated with Akt inhibitor revealed a significant decrease in p-Akt and p-p70S6K that was associated with a significant decrease in vimentin and an increase in N-cadherin expression. In addition, cells transfected with DN-Akt or DN-S6K show significant increase expression in N-cadherin and a decrease in vimentin. Moreover, cells transfected with siRNA against rictor or siRNA against raptor resulted in a decrease in vimentin and an increase N-cadherin expression. Kidney tumors from TSC patients showed significant decrease in N-cadherin and significant increased in vimentin protein expression compared to control kidney tissues. These data comprise the first report to provide the role of Akt/tuberin/mTORC1/2 in the regulation of N-cadherin and vimentin that are involved in the progression of fibrosis in kidney tumor of TSC patients. PMID:25149531

[Effect of DNA polymerase beta on apoptosis and mitochondrial membrane potential induced by hydroquinone, a metabolite of benzene].

PubMed

Chen, Chen; Yang, Mo; Zhang, Zun-zhen; Wu, Mei; Deng, Wen-wen

2011-12-01

To explore the effect and mechanism of DNA polymerase β expression level on cell apoptosis and mitochondrial membrane potential induced by hydroquinone. Polβ wild-type cells (polβ+/+), polβ overexpressed cells (polβ oe) and polβ null cells (polβ-/-) were applied as a model cell system, The effect of cell apoptosis and mitochondrial membrane potential induced by different doses of hydroquinone were analyzed by flow cytometry. The ROS and ·OH assay kit were used to examine the cellular ROS and ·OH level. The activity of cellular SOD and GSH-Px were tested by Chemiluminescence method after exposed to different concentrations of hydroquinone. With the dose of hydroquinone increased, the rate of apoptosis and falling of mitochondrial membrane potential (ΔΨm) in cells were increased compared with the control. When compared with polβ+/+ cells, the rate of apoptosis in polβ-/- cells exposed to 20.00, 40.00, 80.00 µmol/L hydroquinone increased and the rate of apoptosis in polβ oe cells exposed to 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone decreased (P < 0.05). Compared with polβ+/+ cells (20.60% ± 0.57%, 37.95% ± 0.64%, 44.50% ± 1.27%, 57.55% ± 1.06%), the rate of cell which undergone mitochondrial depolarization in polβ-/- cells treated with 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone (33.60% ± 1.55%, 46.05% ± 1.77%, 52.75% ± 2.05%, 75.20% ± 0.56%) increased. The rate of cell which undergone mitochondrial depolarization in polβ oe cells exposed to 10.00, 20.00, 40.00, 80.00 µmol/L hydroquinone (16.05% ± 1.20%, 29.80% ± 1.21%, 35.15% ± 1.06%, 53.80% ± 0.85%) decreased (P < 0.05). When compared with polβ+/+ cells, fluorescent intensity of polβ-/- cells treated with different dosages of hydroquinone increased, while which of polβ oe cells decreased (P < 0.05). Compared with polβ+/+ cells, ·OH level of polβ-/- cells treated with 20.00, 40.00 µmol/L hydroquinone significantly enhanced, while which of polβ oe cells decreased sharply (P < 0.05). Under the same concentrations of hydroquinone, the activity of SOD and GSH-Px were decreased most rapidly in polβ-/- cells. The activity of SOD and GSH-Px in polβ oe cells decreased slower than in the polβ-/- cells. Hydroquinone could induced apoptosis by the generation of ROS and decrease of ΔΨm; polβ could protect cells from apoptosis induced by hydroquinone through decrease of ROS level and depolarization of mitochondria.

Celecoxib and tauro-ursodeoxycholic acid co-treatment inhibits cell growth in familial adenomatous polyposis derived LT97 colon adenoma cells

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

Heumen, Bjorn W.H. van, E-mail: b.vanheumen@mdl.umcn.nl; Roelofs, Hennie M.J.; Morsche, Rene H.M. te

Chemoprevention would be a desirable strategy to avoid duodenectomy in patients with familial adenomatous polyposis (FAP) suffering from duodenal adenomatosis. We investigated the in vitro effects on cell proliferation, apoptosis, and COX-2 expression of the potential chemopreventives celecoxib and tauro-ursodeoxycholic acid (UDCA). HT-29 colon cancer cells and LT97 colorectal micro-adenoma cells derived from a patient with FAP, were exposed to low dose celecoxib and UDCA alone or in combination with tauro-cholic acid (CA) and tauro-chenodeoxycholic acid (CDCA), mimicking bile of FAP patients treated with UDCA. In HT-29 cells, co-treatment with low dose celecoxib and UDCA resulted in a decreased cellmore » growth (14-17%, p < 0.01). A more pronounced decrease (23-27%, p < 0.01) was observed in LT97 cells. Cell growth of HT-29 cells exposed to 'artificial bile' enriched with UDCA, was decreased (p < 0.001), either in the absence or presence of celecoxib. In LT97 cells incubated with 'artificial bile' enriched with UDCA, cell growth was decreased only in the presence of celecoxib (p < 0.05). No clear evidence was found for involvement of proliferating cell nuclear antigen, caspase-3, or COX-2 in the cellular processes leading to the observed changes in cell growth. In conclusion, co-treatment with low dose celecoxib and UDCA has growth inhibitory effects on colorectal adenoma cells derived from a patient with FAP, and further research on this combination as promising chemopreventive strategy is desired. -- Highlights: Black-Right-Pointing-Pointer Celecoxib and UDCA acid co-treatment decreases cell growth in colon tumor cells. Black-Right-Pointing-Pointer UDCA enriched 'artificial bile' decreases LT-97 cell growth only in presence of celecoxib. Black-Right-Pointing-Pointer PCNA, caspase-3, nor COX-2 seem to be involved in the observed changes in cell growth.« less

Spacelab 1 hematology experiment (INS103): Influence of space flight on erythrokinetics in man

NASA Technical Reports Server (NTRS)

Leach, C. S.; Chen, J. P.; Crosby, W.; Dunn, C. D. R.; Johnson, P. C.; Lange, R. D.; Larkin, E.; Tavassoli, M.

1985-01-01

An experiment conducted on the 10-day Spacelab 1 mission aboard the ninth Space Shuttle flight in November to December 1983 was designed to measure factors involved in the control of erythrocyte turnover that might be altered during weightlessness. Blood samples were collected before, during, and after the flight. Immediately after landing, red cell mass showed a mean decrease of 9.3 percent in the four astronauts. Neither hyperoxia nor an increase in blood phosphate was a cause of the decrease. Red cell survival time and iron incorporation postflight were not significantly different from their preflight levels. Serum haptoglobin did not decrease, indicating that intravascular hemolysis was not a major cause of red cell mass change. An increase in serum ferritin after the second day of flight may have been caused by red cell breakdown early in flight. Erythropoietin levels decreased during and after flight, but preflight levels were high and the decrease was not significant. The space flight-induced decrease in red cell mass may result from a failure of erythropoiesis to replace cells destroyed by the spleen soon after weightlessness is attained.

Inhibition of prohormone convertase 1 (PC1) expression in cholecystokinin (CCK) expressing At-T20 cells decreased cellular content and secretion of CCK and caused a shift in molecular forms of CCK secreted.

PubMed

Beinfeld, Margery C; Vishnuvardhan, Daesety; Blum, Alissa; Reynolds, Nicole; Fannous, Sanya; Kitagawa, Kouki; Marchand, James E

2006-04-01

Two different RNAi methods were used to inhibit the expression of prohormone convertase 1 (PC1) in At-T20 cells. Transient transfection of double stranded RNA and stable expression of a vector expressing hairpin-loop RNA targeting PC1 reduced cholecystokinin (CCK) secretion from At-T20 cells. PC1 mRNA and protein were also decreased in the vector transfected cells. This treatment caused a shift in the forms of cholecystokinin (CCK) secreted, decreasing CCK 22 and increasing CCK 8. Stable expression of RNAi effectively decreased PC1 expression. The observed decrease in CCK seen with these RNAi treatments further supports a role for PC1 in CCK processing in these cells.

Radiation and temperature effects in gallium arsenide, indium phosphide and silicon solar cells

NASA Technical Reports Server (NTRS)

Weinberg, I.; Swartz, C. K.; Hart, R. E., Jr.; Statler, R. L.

1987-01-01

The effects of radiation on performance are determined for both n(+)p and p(+)n GaAs and InP cells and for silicon n(+)p cells. It is found that the radiation resistance of InP is greater than that of both GaAs and Si under 1 MeV electron irradiation. For silicon, the observed decreased radiation resistance with decreased resistivity is attributed to the presence of a radiation induced boron-oxygen defect. Comparison of radiation damage in both p(+)n and n(+)p GaAs cells yields a decreased radiation resistance for the n(+)p cell attributable to increased series resistance, decreased shunt resistance, and relatively greater losses in the cell's p-region. For InP, the n(+)p configuration is found to have greater radiation resistance than the p(+)n cell. The increased loss in this latter cell is attributed to losses in the cell's emitter region. Temperature dependency results are interpreted using a theoretical relation for dVoc/cT which predicts that increased Voc should results in decreased numerical values for dPm/dT. The predicted correlation is observed for GaAs but not for InP a result which is attributed to variations in cell processing.

Radiation and temperature effects in gallium arsenide, indium phosphide, and silicon solar cells

NASA Technical Reports Server (NTRS)

Weinberg, I.; Swartz, C. K.; Hart, R. E., Jr.; Statler, R. L.

1987-01-01

The effects of radiation on performance are determined for both n+p and p+n GaAs and InP cells and for silicon n+p cells. It is found that the radiation resistance of InP is greater than that of both GaAs and Si under 1-MeV electron irradiation. For silicon, the observed decreased radiation resistance with decreased resistivity is attributed to the presence of a radiation-induced boron-oxygen defect. Comparison of radiation damage in both p+n and n+p GaAs cells yields a decreased radiation resistance for the n+p cell attributable to increased series resistance, decreased shunt resistance, and relatively greater losses in the cell's p-region. For InP, the n+p configuration is found to have greater radiation resistance than the p+n cell. The increased loss in this latter cell is attributed to losses in the cell's emitter region. Temperature dependency results are interpreted using a theoretical relation for dVoc/dT, which predicts that increased Voc should result in decreased numerical values for dPm/dT. The predicted correlation is observed for GaAs but not for InP, a result which is attributed to variations in cell processing.

Corneal epithelial wound healing and bactericidal effect of conditioned medium from human uterine cervical stem cells.

PubMed

Bermudez, Maria A; Sendon-Lago, Juan; Eiro, Noemi; Treviño, Mercedes; Gonzalez, Francisco; Yebra-Pimentel, Eva; Giraldez, Maria Jesus; Macia, Manuel; Lamelas, Maria Luz; Saa, Jorge; Vizoso, Francisco; Perez-Fernandez, Roman

2015-01-22

To evaluate the effect of conditioned medium from human uterine cervical stem cells (CM-hUCESCs) on corneal epithelial healing in a rat model of dry eye after alkaline corneal epithelial ulcer. We also tested the bactericidal effect of CM-hUCESCs. Dry eye was induced in rats by extraocular lacrimal gland excision, and corneal ulcers were produced using NaOH. Corneal histologic evaluation was made with hematoxylin-eosin (H&E) staining. Real-time PCR was used to evaluate mRNA expression levels of proinflammatory cytokines. We also studied the bactericidal effect of CM-hUCESCs in vitro and on infected corneal contact lenses (CLs) using Escherichia coli and Staphylococcus epidermidis bacteria. In addition, in order to investigate proteins from CM-hUCESCs that could mediate these effects, we carried out a human cytokine antibody array. After injury, dry eyes treated with CM-hUCESCs significantly improved epithelial regeneration and showed reduced corneal macrophage inflammatory protein-1 alpha (MIP-1α) and TNF-α mRNA expression as compared to untreated eyes and eyes treated with culture medium or sodium hyaluronate ophthalmic drops. In addition, we found in CM-hUCESCs high levels of proteins, such as tissue inhibitors of metalloproteinases 1 and 2, fibroblast growth factor 6 and 7, urokinase receptor, and hepatocyte growth factor, that could mediate these effects. In vitro, CM-hUCESCs showed a clear bactericidal effect on both E. coli and S. epidermidis and CLs infected with S. epidermidis. Analyses of CM-hUCESCs showed elevated levels of proteins that could be involved in the bactericidal effect, such as the chemokine (C-X-C motif) ligands 1, 6, 8, 10, and the chemokine (C-C motif) ligands 5 and 20. Treatment with CM-hUCESCs improved wound healing of alkali-injured corneas and showed a strong bactericidal effect on CLs. Patients using CLs and suffering from dry eye, allergies induced by commercial solutions, or small corneal injuries could benefit from this treatment. Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.

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.

The Effects of Oxygen Level and Glucose Concentration on the Metabolism of Porcine TMJ Disc Cells

PubMed Central

Cisewski, Sarah E.; Zhang, Lixia; Kuo, Jonathan; Wright, Gregory J.; Wu, Yongren; Kern, Michael J.; Yao, Hai

2015-01-01

Objective To determine the combined effect of oxygen level and glucose concentration on cell viability, ATP production, and matrix synthesis of temporomandibular joint (TMJ) disc cells. Design TMJ disc cells were isolated from pigs aged 6-8 months and cultured in a monolayer. Cell cultures were preconditioned for 48 hours with 0, 1.5, 5, or 25mM glucose DMEM under 1%, 5%, 10%, or 21% O2 level, respectively. The cell viability was measured using the WST-1 assay. ATP production was determined using the Luciferin-Luciferase assay. Collagen and proteoglycan synthesis were determined by measuring the incorporation of [2, 3-3H]proline and [35S]sulfate into the cells, respectively. Results TMJ disc cell viability significantly decreased (P<0.0001) without glucose. With glucose present, decreased oxygen levels significantly increased viability (P<0.0001), while a decrease in glucose concentration significantly decreased viability (P<0.0001). With glucose present, decreasing oxygen levels significantly reduced ATP production (P<0.0001) and matrix synthesis (P<0.0001). A decreased glucose concentration significantly decreased collagen synthesis (P<0.0001). The interaction between glucose and oxygen was significant in regards to cell viability (P<0.0001), ATP production (P=0.00015), and collagen (P=0.0002) and proteoglycan synthesis (P<0.0001). Conclusions Although both glucose and oxygen are important, glucose is the limiting nutrient for TMJ disc cell survival. At low oxygen levels, the production of ATP, collagen, and proteoglycan are severely inhibited. These results suggest that steeper nutrient gradients may exist in the TMJ disc and it may be vulnerable to pathological events that impede nutrient supply. PMID:26033165

The effects of oxygen level and glucose concentration on the metabolism of porcine TMJ disc cells.

PubMed

Cisewski, S E; Zhang, L; Kuo, J; Wright, G J; Wu, Y; Kern, M J; Yao, H

2015-10-01

To determine the combined effect of oxygen level and glucose concentration on cell viability, ATP production, and matrix synthesis of temporomandibular joint (TMJ) disc cells. TMJ disc cells were isolated from pigs aged 6-8 months and cultured in a monolayer. Cell cultures were preconditioned for 48 h with 0, 1.5, 5, or 25 mM glucose DMEM under 1%, 5%, 10%, or 21% O2 level, respectively. The cell viability was measured using the WST-1 assay. ATP production was determined using the Luciferin-Luciferase assay. Collagen and proteoglycan synthesis were determined by measuring the incorporation of [2, 3-(3)H] proline and [(35)S] sulfate into the cells, respectively. TMJ disc cell viability significantly decreased (P < 0.0001) without glucose. With glucose present, decreased oxygen levels significantly increased viability (P < 0.0001), while a decrease in glucose concentration significantly decreased viability (P < 0.0001). With glucose present, decreasing oxygen levels significantly reduced ATP production (P < 0.0001) and matrix synthesis (P < 0.0001). A decreased glucose concentration significantly decreased collagen synthesis (P < 0.0001). The interaction between glucose and oxygen was significant in regards to cell viability (P < 0.0001), ATP production (P = 0.00015), and collagen (P = 0.0002) and proteoglycan synthesis (P < 0.0001). Although both glucose and oxygen are important, glucose is the limiting nutrient for TMJ disc cell survival. At low oxygen levels, the production of ATP, collagen, and proteoglycan are severely inhibited. These results suggest that steeper nutrient gradients may exist in the TMJ disc and it may be vulnerable to pathological events that impede nutrient supply. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Exercise training improves obesity‐related lymphatic dysfunction

PubMed Central

Hespe, Geoffrey E.; Kataru, Raghu P.; Savetsky, Ira L.; García Nores, Gabriela D.; Torrisi, Jeremy S.; Nitti, Matthew D.; Gardenier, Jason C.; Zhou, Jie; Yu, Jessie Z.; Jones, Lee W.

2016-01-01

Key points Obesity results in perilymphatic inflammation and lymphatic dysfunction.Lymphatic dysfunction in obesity is characterized by decreased lymphatic vessel density, decreased collecting lymphatic vessel pumping frequency, decreased lymphatic trafficking of immune cells, increased lymphatic vessel leakiness and changes in the gene expression patterns of lymphatic endothelial cells.Aerobic exercise, independent of weight loss, decreases perilymphatic inflammatory cell accumulation, improves lymphatic function and reverses pathological changes in gene expression in lymphatic endothelial cells. Abstract Although previous studies have shown that obesity markedly decreases lymphatic function, the cellular mechanisms that regulate this response remain unknown. In addition, it is unclear whether the pathological effects of obesity on the lymphatic system are reversible with behavioural modifications. The purpose of this study, therefore, was to analyse lymphatic vascular changes in obese mice and to determine whether these pathological effects are reversible with aerobic exercise. We randomized obese mice to either aerobic exercise (treadmill running for 30 min per day, 5 days a week, for 6 weeks) or a sedentary group that was not exercised and analysed lymphatic function using a variety of outcomes. We found that sedentary obese mice had markedly decreased collecting lymphatic vessel pumping capacity, decreased lymphatic vessel density, decreased lymphatic migration of immune cells, increased lymphatic vessel leakiness and decreased expression of lymphatic specific markers compared with lean mice (all P < 0.01). Aerobic exercise did not cause weight loss but markedly improved lymphatic function compared with sedentary obese mice. Exercise had a significant anti‐inflammatory effect, resulting in decreased perilymphatic accumulation of inflammatory cells and inducible nitric oxide synthase expression. In addition, exercise normalized isolated lymphatic endothelial cell gene expression of lymphatic specific genes, including VEGFR‐3 and Prox1. Taken together, our findings suggest that obesity impairs lymphatic function via multiple mechanisms and that these pathological changes can be reversed, in part, with aerobic exercise, independent of weight loss. In addition, our study shows that obesity‐induced lymphatic endothelial cell gene expression changes are reversible with behavioural modifications. PMID:26931178

Effect of tributyltin (TBT) on ATP levels in human natural killer (NK) cells: relationship to TBT-induced decreases in NK function.

PubMed

Dudimah, Fred D; Odman-Ghazi, Sabah O; Hatcher, Frank; Whalen, Margaret M

2007-01-01

The purpose of this study was to investigate the role that tributyltin (TBT)-induced decreases in ATP levels may play in TBT-induced decreases in the tumor lysing (lytic) function of natural killer (NK) cells. NK cells are a subset of lymphocytes that act as an initial immune defense against tumor cells and virally infected cells. TBT is an environmental contaminant that has been detected in human blood, which has been shown to interfere with ATP synthesis. Previous studies have shown that TBT is able to decrease very significantly the lytic function of NK cells. In this study NK cells were exposed to various concentrations of TBT and to two other compounds that interfere with ATP synthesis (rotenone a complex I inhibitor and oligomycin an ATP synthase inhibitor) for various lengths of time before determining the levels of ATP and lytic function. Exposures of NK cells to 10, 25, 50 and 100 nm TBT did not significantly reduce ATP levels after 24 h. However, these same exposures caused significant decreases in cytotoxic function. Studies of brief 1 h exposures to a range of TBT, rotenone and oligomycin concentrations followed by 24 h, 48 h and 6 day periods in compound-free media prior to assaying for ATP levels or cytotoxic function showed that each of the compounds caused persistent decreases in ATP levels and lytic function of NK cells. Exposures to 0.05-5 microm rotenone or oligomycin for 1 h reduced ATP levels by 20-25% but did not have any measurable effect on the ability of NK cells to lyse tumor cells. ATP levels were also decreased by about 20-25% after 24 h or 48 h exposures to rotenone or oligomycin (0.5 microm ), and the lytic function was decreased by about 50%. The results suggest that TBT-induced decreases in ATP levels were not responsible for the loss of cytotoxic function seen at 1 h and 24 h. However, TBT-induced decreases of NK-ATP levels may be at least in part responsible for losses of NK-cytotoxic function seen after 48 h and 6 day exposures. Copyright 2006 John Wiley & Sons, Ltd.

NaK-ATPase pump sites in cultured bovine corneal endothelium of varying cell density at confluence.

PubMed

Crawford, K M; Ernst, S A; Meyer, R F; MacCallum, D K

1995-06-01

The driving force for ion and water flow necessary for efficient deturgesence of the corneal stroma resides in the ouabain-sensitive sodium (Na) pump of corneal endothelial cells. Using a cell culture model of corneal endothelial cell hypertrophy, the authors examined the expression of Na pumps at the cell surface to see how this central element of the endothelial pump changed as corneal endothelial cell density decreased to a level associated with corneal decompensation in vivo. 3H-ouabain binding to NaK-ATPase at saturating conditions was used to quantitate the number of Na pump sites on cultured bovine corneal endothelial cells as the confluent density decreased from approximately 2750 cells/mm2 to approximately 275 cells/mm2. The mean number of Na pump sites per cell at confluence (1.92 +/- 0.07 x 10(6)) did not change as the cell density decreased 2.7-fold from 2763 cells/mm2 to 1000 cells/mm2. However, pump site expression doubled to approximately 4 x 10(6) sites/cell as the cell density decreased from 1000 cells/mm2 to 275 cells/mm2. Despite the incremental increase in Na pump site expression that occurred as the cells hypertrophied below a density of 1000/mm2 to achieve confluence, this increase was insufficient to prevent a decrease in Na pump site density of the intact monolayer, expressed as pump sites/mm2. The confluent cell density of cultured bovine corneal endothelial cells can be varied from that found in the normal native cornea to that associated with corneal decompensation. In confluent cultures with cell densities ranging from 2750 cells/mm2 to 1000 cells/mm2, the number of pump sites per cell remains relatively unchanged. Below cell densities of 1000 cells/mm2, the number of pump sites per cell progressively increases. The increased Na pump site abundance in markedly hypertrophied endothelial cells cannot adequately compensate for the progressive reduction in the number of transporting cells per unit area within the intact monolayer. Even when considered with the decrease in the size of the paracellular ion conductive pathway that is a consequence of progressive endothelial hypertrophy, the overall pumping capacity of the intact endothelial monolayer declines.

Effects of first-line chemotherapy on natural killer cells in adult T-cell leukemia-lymphoma and peripheral T-cell lymphoma.

PubMed

Ogura, Michinori; Ishida, Takashi; Tsukasaki, Kunihiro; Takahashi, Takeshi; Utsunomiya, Atae

2016-07-01

Natural killer (NK) cells are well known to be the most important effector cells mediating antibody-dependent cellular cytotoxicity (ADCC) which is an important mechanism of action of antibody drugs. We evaluated the effects of chemotherapy on the cell number and activity of NK cells from patients who received the vincristine-cyclophosphamide-doxorubicin-prednisone (VCAP), doxorubicin-ranimustine-prednisone (AMP), and vindesine-etoposide-carboplatin-prednisone (VECP) (mLSG15) or mLSG15-like (-L) regimen, which is one of the standard of cares for newly diagnosed adult T-cell leukemia-lymphoma (ATL), or the cyclophosphamide-doxorubicin-vincristine-prednisone (CHOP) or CHOP-L regimen which is another standard of care for ATL and peripheral T-cell lymphoma (PTCL). The number of lymphocytes and NK cells, and NK cell activity, were assessed using flow cytometry and a (51)Cr release assay, respectively. A total of 26 patients with untreated ATL or PTCL were enrolled, and blood samples from 25 patients were evaluable. NK cell number in ATL decreased after mLSG15/-L treatment, and the degree of decrease in the NK cell number was more prominent just before VECP therapy (Day 15-17 of each cycle) than just before VCAP therapy (Day 1 of each cycle). The NK cell number in ATL after CHOP/-L treatment also decreased. Interestingly, the NK cell activity showed a tendency to increase after the treatment. NK cell number in PTCL did not decrease by CHOP/-L regimen, but the activity was slightly decreased after the treatment. These results indicate that the effects of chemotherapeutic agents on NK cells vary according to the disease type and intensity of chemotherapy.

Bone marrow support of the heart in pressure overload is lost with aging.

PubMed

Sopko, Nikolai A; Turturice, Benjamin A; Becker, Mitchell E; Brown, Chase R; Dong, Feng; Popović, Zoran B; Penn, Marc S

2010-12-21

Exogenous stem cell delivery is under investigation to prevent and treat cardiac dysfunction. It is less studied as to the extent endogenous bone marrow derived stem cells contribute to cardiac homeostais in response to stress and the affects of aging on this stress response. To determine the role of bone marrow (BM) derived stem cells on cardiac homeostasis in response to pressure overload (PO) and how this response is altered by aging. Young (8 weeks) and old (>40 weeks) C57/b6 mice underwent homo- and heterochronic BM transplantation prior to transverse aortic constriction (TAC). We found that older BM is associated with decreased cardiac function following TAC. This decreased function is associated with decrease in BM cell engraftment, increased myocyte apoptosis, decreased myocyte hypertrophy, increased myocardial fibrosis and decreased cardiac function. Additionally, there is a decrease in activation of resident cells within the heart in response to PO in old mice. Interestingly, these effects are not due to alterations in vascular density or inflammation in response to PO or differences in ex vivo stem cell migration between young and old mice. BM derived stem cells are activated in response to cardiac PO, and the recruitment of BM derived cells are involved in cardiac myocyte hypertrophy and maintenance of function in response to PO which is lost with aging.

Decreased IL-10 production mediated by Toll-like receptor 9 in B cells in multiple sclerosis.

PubMed

Hirotani, Makoto; Niino, Masaaki; Fukazawa, Toshiyuki; Kikuchi, Seiji; Yabe, Ichiro; Hamada, Shinsuke; Tajima, Yasutaka; Sasaki, Hidenao

2010-04-15

The complexity of the roles of Toll-like receptors (TLRs) is attributable to their ability to promote or suppress autoimmune diseases. Recent studies have demonstrated that B cells regulate autoimmune diseases, including multiple sclerosis (MS), by producing interleukin (IL)-10. By using CpG DNA as a TLR9 agonist, we investigated the immunoregulatory functions of B cell via TLR9 in MS. Our results indicate that TLR9-mediated IL-10 production by B cells was significantly decreased in MS, and this decrease is likely due to decreased TLR9 expression in memory B cells, suggesting a role of TLR9 in immunoregulation in MS. Copyright 2010 Elsevier B.V. All rights reserved.

Inhibition of Regulatory Volume Decrease Enhances the Cytocidal Effect of Hypotonic Shock in Hepatocellular Carcinoma.

PubMed

Kudou, Michihiro; Shiozaki, Atsushi; Kosuga, Toshiyuki; Ichikawa, Daisuke; Konishi, Hirotaka; Morimura, Ryo; Komatsu, Shuhei; Ikoma, Hisashi; Fujiwara, Hitoshi; Okamoto, Kazuma; Hosogi, Shigekuni; Nakahari, Takashi; Marunaka, Yoshinori; Otsuji, Eigo

2016-01-01

Background : Hypotonic shock induces cytocidal effects through cell rupture, and cancer therapy based on this mechanism has been clinically administered to hepatocellular carcinoma patients. We herein investigated the effectiveness of hypotonic shock combined with the inhibition of regulatory volume decrease as cancer therapy for hepatocellular carcinoma. Methods : Morphological changes in human hepatocellular carcinoma cell lines were observed under a differential interference contrast microscope connected to a high-speed digital video camera. Cell volume changes under hypotonic shock with or without chloride, potassium, or water channel blockers were observed using a high-resolution flow cytometer. In order to investigate cytocidal effects, the number of surviving cells was compared after exposure to hypotonic solution with and without each channel blocker (re-incubation experiment). Results : Video recordings showed that cells exposed to distilled water rapidly swelled and then ruptured. Cell volume measurements revealed regulatory volume decrease under mild hypotonic shock, whereas severe hypotonic shock increased the number of broken fragments as a result of cell rupture. Moreover, regulatory volume decrease was inhibited in cells treated with each channel blocker. Re-incubation experiments showed the cytocidal effects of hypotonic shock in cells exposed to hypotonic solution, and additional treatments with each channel blocker enhanced these effects. Conclusion : The inhibition of regulatory volume decrease with chloride, potassium, or water channel blockers may enhance the cytocidal effects of hypotonic shock in hepatocellular carcinoma. Hypotonic shock combined with the inhibition of regulatory volume decrease was a more effective therapy than hypotonic shock alone.

Inhibition of Regulatory Volume Decrease Enhances the Cytocidal Effect of Hypotonic Shock in Hepatocellular Carcinoma

PubMed Central

Kudou, Michihiro; Shiozaki, Atsushi; Kosuga, Toshiyuki; Ichikawa, Daisuke; Konishi, Hirotaka; Morimura, Ryo; Komatsu, Shuhei; Ikoma, Hisashi; Fujiwara, Hitoshi; Okamoto, Kazuma; Hosogi, Shigekuni; Nakahari, Takashi; Marunaka, Yoshinori; Otsuji, Eigo

2016-01-01

Background: Hypotonic shock induces cytocidal effects through cell rupture, and cancer therapy based on this mechanism has been clinically administered to hepatocellular carcinoma patients. We herein investigated the effectiveness of hypotonic shock combined with the inhibition of regulatory volume decrease as cancer therapy for hepatocellular carcinoma. Methods: Morphological changes in human hepatocellular carcinoma cell lines were observed under a differential interference contrast microscope connected to a high-speed digital video camera. Cell volume changes under hypotonic shock with or without chloride, potassium, or water channel blockers were observed using a high-resolution flow cytometer. In order to investigate cytocidal effects, the number of surviving cells was compared after exposure to hypotonic solution with and without each channel blocker (re-incubation experiment). Results: Video recordings showed that cells exposed to distilled water rapidly swelled and then ruptured. Cell volume measurements revealed regulatory volume decrease under mild hypotonic shock, whereas severe hypotonic shock increased the number of broken fragments as a result of cell rupture. Moreover, regulatory volume decrease was inhibited in cells treated with each channel blocker. Re-incubation experiments showed the cytocidal effects of hypotonic shock in cells exposed to hypotonic solution, and additional treatments with each channel blocker enhanced these effects. Conclusion: The inhibition of regulatory volume decrease with chloride, potassium, or water channel blockers may enhance the cytocidal effects of hypotonic shock in hepatocellular carcinoma. Hypotonic shock combined with the inhibition of regulatory volume decrease was a more effective therapy than hypotonic shock alone. PMID:27471568

Vaccinia virus decreases major histocompatibility complex (MHC) class II antigen presentation, T-cell priming, and peptide association with MHC class II

PubMed Central

Rehm, Kristina E; Connor, Ramsey F; Jones, Gwendolyn J B; Yimbu, Kenneth; Mannie, Mark D; Roper, Rachel L

2009-01-01

Vaccinia virus (VACV) is the current live virus vaccine used to protect humans against smallpox and monkeypox, but its use is contraindicated in several populations because of its virulence. It is therefore important to elucidate the immune evasion mechanisms of VACV. We found that VACV infection of antigen-presenting cells (APCs) significantly decreased major histocompatibility complex (MHC) II antigen presentation and decreased synthesis of 13 chemokines and cytokines, suggesting a potent viral mechanism for immune evasion. In these model systems, responding T cells were not directly affected by virus, indicating that VACV directly affects the APC. VACV significantly decreased nitric oxide production by peritoneal exudate cells and the RAW macrophage cell line in response to lipopolysaccharide (LPS) and interferon (IFN)-γ, decreased class II MHC expression on APCs, and induced apoptosis in macrophages and dendritic cells. However, VACV decreased antigen presentation by 1153 B cells without apparent apoptosis induction, indicating that VACV differentially affects B lymphocytes and other APCs. We show that the key mechanism of VACV inhibition of antigen presentation may be its reduction of antigenic peptide loaded into the cleft of MHC class II molecules. These data indicate that VACV evades the host immune response by impairing critical functions of the APC. PMID:20067538