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Sample records for activator inhibitor pai-1

  1. Genetics of Plasminogen Activator Inhibitor-1 (PAI-1) in a Ghanaian Population

    PubMed Central

    White, Marquitta J.; Kodaman, Nuri M.; Harder, Reed H.; Asselbergs, Folkert W.; Vaughan, Douglas E.; Brown, Nancy J.; Moore, Jason H.; Williams, Scott M.

    2015-01-01

    Plasminogen activator inhibitor 1 (PAI-1), a major modulator of the fibrinolytic system, is an important factor in cardiovascular disease (CVD) susceptibility and severity. PAI-1 is highly heritable, but the few genes associated with it explain only a small portion of its variation. Studies of PAI-1 typically employ linear regression to estimate the effects of genetic variants on PAI-1 levels, but PAI-1 is not normally distributed, even after transformation. Therefore, alternative statistical methods may provide greater power to identify important genetic variants. Additionally, most genetic studies of PAI-1 have been performed on populations of European descent, limiting the generalizability of their results. We analyzed >30,000 variants for association with PAI-1 in a Ghanaian population, using median regression, a non-parametric alternative to linear regression. Three variants associated with median PAI-1, the most significant of which was in the gene arylsulfatase B (ARSB) (p = 1.09 x 10−7). We also analyzed the upper quartile of PAI-1, the most clinically relevant part of the distribution, and found 19 SNPs significantly associated in this quartile. Of note an association was found in period circadian clock 3 (PER3). Our results reveal novel associations with median and elevated PAI-1 in an understudied population. The lack of overlap between the two analyses indicates that the genetic effects on PAI-1 are not uniform across its distribution. They also provide evidence of the generalizability of the circadian pathway’s effect on PAI-1, as a recent meta-analysis performed in Caucasian populations identified another circadian clock gene (ARNTL). PMID:26322636

  2. Genetics of Plasminogen Activator Inhibitor-1 (PAI-1) in a Ghanaian Population.

    PubMed

    White, Marquitta J; Kodaman, Nuri M; Harder, Reed H; Asselbergs, Folkert W; Vaughan, Douglas E; Brown, Nancy J; Moore, Jason H; Williams, Scott M

    2015-01-01

    Plasminogen activator inhibitor 1 (PAI-1), a major modulator of the fibrinolytic system, is an important factor in cardiovascular disease (CVD) susceptibility and severity. PAI-1 is highly heritable, but the few genes associated with it explain only a small portion of its variation. Studies of PAI-1 typically employ linear regression to estimate the effects of genetic variants on PAI-1 levels, but PAI-1 is not normally distributed, even after transformation. Therefore, alternative statistical methods may provide greater power to identify important genetic variants. Additionally, most genetic studies of PAI-1 have been performed on populations of European descent, limiting the generalizability of their results. We analyzed >30,000 variants for association with PAI-1 in a Ghanaian population, using median regression, a non-parametric alternative to linear regression. Three variants associated with median PAI-1, the most significant of which was in the gene arylsulfatase B (ARSB) (p = 1.09 x 10(-7)). We also analyzed the upper quartile of PAI-1, the most clinically relevant part of the distribution, and found 19 SNPs significantly associated in this quartile. Of note an association was found in period circadian clock 3 (PER3). Our results reveal novel associations with median and elevated PAI-1 in an understudied population. The lack of overlap between the two analyses indicates that the genetic effects on PAI-1 are not uniform across its distribution. They also provide evidence of the generalizability of the circadian pathway's effect on PAI-1, as a recent meta-analysis performed in Caucasian populations identified another circadian clock gene (ARNTL).

  3. Modulation of plasminogen activator inhibitor-1 (PAI-1) by the naphthoquinone shikonin.

    PubMed

    Han, Tingting; Zhang, Guangping; Yan, Dong; Yang, Hong; Ma, Tonghui; Ye, Zuguang

    2016-09-01

    Plasminogen activator inhibitor-1 (PAI-1) is a key negative regulator of the fibrinolytic system. Elevated levels of PAI-1 are associated with thrombosis and cardiovascular and metabolic diseases. Inhibition of PAI-1 activity represents a new strategy for antithrombotic and antifibrinolysis therapies. In this study, we systematically investigated the inhibitory effect of shikonin on PAI-1 activity. In the chromogenic substrate-based urokinase (uPA)/PAI-1 assay, we found that shikonin inhibited human PAI-1 activity with IC50 values of 30.68±2.32μM. This result was further confirmed by urokinase-type plasminogen activator (uPA)-mediated clot lysis assay. Mechanistic studies indicated that shikonin directly could bind to PAI-1 and prevent the binding of PAI-1 to uPA in a dose-dependent manner. Shikonin also blocked the formation of PAI-1/uPA complex, as shown by SDS/PAGE analysis. In the mouse arterial thrombosis model, intraperitoneal injection of shikonin at 1mgkg(-1) dose significantly prolonged tail bleeding time from 12.956±4.457min to 26.576±2.443min. It also reduced arterial thrombus weight from 0.01±0.001g to 0.006±0.001g (p<0.05). In a liver fibrosis treatment model, when shikonin was continuously injected intraperitoneally at a dose of 1mgkg(-1) over a two-week period, the hydroxyproline content in the mice plasma was significantly reduced and the degree of liver fibrosis was decreased significantly. Thus, shikonin may represent a novel small molecule inhibitor of PAI-1 that could have become a lead drug the treatment of thrombus and fibrosis. PMID:27476618

  4. Modulation of plasminogen activator inhibitor-1 (PAI-1) by the naphthoquinone shikonin.

    PubMed

    Han, Tingting; Zhang, Guangping; Yan, Dong; Yang, Hong; Ma, Tonghui; Ye, Zuguang

    2016-09-01

    Plasminogen activator inhibitor-1 (PAI-1) is a key negative regulator of the fibrinolytic system. Elevated levels of PAI-1 are associated with thrombosis and cardiovascular and metabolic diseases. Inhibition of PAI-1 activity represents a new strategy for antithrombotic and antifibrinolysis therapies. In this study, we systematically investigated the inhibitory effect of shikonin on PAI-1 activity. In the chromogenic substrate-based urokinase (uPA)/PAI-1 assay, we found that shikonin inhibited human PAI-1 activity with IC50 values of 30.68±2.32μM. This result was further confirmed by urokinase-type plasminogen activator (uPA)-mediated clot lysis assay. Mechanistic studies indicated that shikonin directly could bind to PAI-1 and prevent the binding of PAI-1 to uPA in a dose-dependent manner. Shikonin also blocked the formation of PAI-1/uPA complex, as shown by SDS/PAGE analysis. In the mouse arterial thrombosis model, intraperitoneal injection of shikonin at 1mgkg(-1) dose significantly prolonged tail bleeding time from 12.956±4.457min to 26.576±2.443min. It also reduced arterial thrombus weight from 0.01±0.001g to 0.006±0.001g (p<0.05). In a liver fibrosis treatment model, when shikonin was continuously injected intraperitoneally at a dose of 1mgkg(-1) over a two-week period, the hydroxyproline content in the mice plasma was significantly reduced and the degree of liver fibrosis was decreased significantly. Thus, shikonin may represent a novel small molecule inhibitor of PAI-1 that could have become a lead drug the treatment of thrombus and fibrosis.

  5. Neutralization of plasminogen activator inhibitor I (PAI-1) by the synthetic antagonist PAI-749 via a dual mechanism of action.

    PubMed

    Gardell, Stephen J; Krueger, Julie A; Antrilli, Thomas A; Elokdah, Hassan; Mayer, Scott; Orcutt, Steven J; Crandall, David L; Vlasuk, George P

    2007-10-01

    PAI-749 is a potent and selective synthetic antagonist of plasminogen activator inhibitor 1 (PAI-1) that preserved tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) activities in the presence of PAI-1 (IC(50) values, 157 and 87 nM, respectively). The fluorescence (Fl) of fluorophore-tagged PAI-1 (PAI-NBD119) was quenched by PAI-749; the apparent K(d) (254 nM) was similar to the IC(50) (140 nM) for PAI-NBD119 inactivation. PAI-749 analogs displayed the same potency rank order for neutralizing PAI-1 activity and perturbing PAI-NBD119 Fl; hence, binding of PAI-749 to PAI-1 and inactivation of PAI-1 activity are tightly linked. Exposure of PAI-1 to PAI-749 for 5 min (sufficient for full inactivation) followed by PAI-749 sequestration with Tween 80 micelles yielded active PAI-1; thus, PAI-749 did not irreversibly inactivate PAI-1, a known metastable protein. Treatment of PAI-1 with a PAI-749 homolog (producing less assay interference) blocked the ability of PAI-1 to displace p-aminobenzamidine from the uPA active site. Consistent with this observation, PAI-749 abolished formation of the SDS-stable tPA/PAI-1 complex. PAI-749-mediated neutralization of PAI-1 was associated with induction of PAI-1 polymerization as assessed by native gel electrophoresis. PAI-749 did not turn PAI-1 into a substrate for tPA; however, PAI-749 promoted plasmin-mediated degradation of PAI-1. In conclusion, PAI-1 inactivation by PAI-749 using purified components can result from a dual mechanism of action. First, PAI-749 binds directly to PAI-1, blocks PAI-1 from accessing the active site of tPA, and abrogates formation of the SDS-stable tPA/PAI-1 complex. Second, binding of PAI-749 to PAI-1 renders PAI-1 vulnerable to plasmin-mediated proteolytic degradation. PMID:17622579

  6. The regulatory region of the human plasminogen activator inhibitor type-1 (PAI-1) gene.

    PubMed Central

    Riccio, A; Lund, L R; Sartorio, R; Lania, A; Andreasen, P A; Danø, K; Blasi, F

    1988-01-01

    The human gene for plasminogen activator inhibitor type-1 (PAI-1) has been isolated and its promoter region characterized. PAI-1 regulation by glucocorticoids, transforming growth factor-beta (TGF-beta) and the phorbol ester PMA is shown to be exerted at the promoter level. A fragment spanning 805 nucleotides of the 5' flanking and 72 of the 5' untranslated region contain information enough to promote transcription and to respond to glucocorticoids when fused to a reporter gene and transfected into human fibrosarcoma cells. A moderately repetitive DNA sequence, containing a TATA box, a GRE consensus, a Z-DNA forming sequence and two imperfect direct repeats at the extremities, is present a few nucleotides 5' of the human PAI-1 gene transcription start site, raising the possibility that this gene could have been activated by DNA insertion during evolution. Images PMID:3130610

  7. Genetic association of five plasminogen activator inhibitor-1 (PAI-1) polymorphisms and idiopathic recurrent pregnancy loss in Korean women.

    PubMed

    Jeon, Young Joo; Kim, Young Ran; Lee, Bo Eun; Choi, Yi Seul; Kim, Ji Hyang; Shin, Ji Eun; Rah, HyungChul; Cha, Sun Hee; Lee, Woo Sik; Kim, Nam Keun

    2013-10-01

    Plasminogen activator inhibitor-1 (PAI-1) is important for maintaining pregnancy. Aberrantly increased PAI-1 levels may contribute to thrombosis and inflammation, leading to pregnancy loss. This study investigated the association of PAI-1 polymorphisms (PAI-1 rs2227631 [-844G>A], rs1799889 [-675 4G/5G], rs6092 [43G>A], rs2227694 [9785G>A], and rs7242 [11053T>G]) with idiopathic recurrent pregnancy loss (RPL) in Korean women. We screened 308 RPL patients and 227 control participants for five PAI-1 polymorphisms. Genotyping of PAI-1 was performed by polymerase chain reaction-restriction fragment length polymorphism assay. PAI-1 4G4G and -844AA/4G4G/11053GG genotypes were associated with RPL. PAI-1 -844A/4G/43G/9785G/11053G haplotype was connected to hypofibrinolytic status (i.e. increased levels of plasma PAI-1, increased numbers of platelets, reduced prothrombin time, and reduced activated partial thromboplastin time). Moreover, PAI-1 11053TG+GG frequency was positively related to plasma homocysteine and urate levels, whereas -844AA frequency was associated with plasma folate concentrations according to ordinal logistic regression analysis. Based on these results, we propose that PAI-1 -844G>A, 4G/5G, and 11053T>G polymorphisms are markers of RPL.

  8. Glioma-derived plasminogen activator inhibitor-1 (PAI-1) regulates the recruitment of LRP1 positive mast cells.

    PubMed

    Roy, Ananya; Coum, Antoine; Marinescu, Voichita D; Põlajeva, Jelena; Smits, Anja; Nelander, Sven; Uhrbom, Lene; Westermark, Bengt; Forsberg-Nilsson, Karin; Pontén, Fredrik; Tchougounova, Elena

    2015-09-15

    Glioblastoma (GBM) is a high-grade glioma with a complex microenvironment, including various inflammatory cells and mast cells (MCs) as one of them. Previously we had identified glioma grade-dependent MC recruitment. In the present study we investigated the role of plasminogen activator inhibitor 1 (PAI-1) in MC recruitment.PAI-1, a primary regulator in the fibrinolytic cascade is capable of forming a complex with fibrinolytic system proteins together with low-density lipoprotein receptor-related protein 1 (LRP1). We found that neutralizing PAI-1 attenuated infiltration of MCs. To address the potential implication of LRP1 in this process, we used a LRP1 antagonist, receptor-associated protein (RAP), and demonstrated the attenuation of MC migration. Moreover, a positive correlation between the number of MCs and the level of PAI-1 in a large cohort of human glioma samples was observed. Our study demonstrated the expression of LRP1 in human MC line LAD2 and in MCs in human high-grade glioma. The activation of potential PAI-1/LRP1 axis with purified PAI-1 promoted increased phosphorylation of STAT3 and subsequently exocytosis in MCs.These findings indicate the influence of the PAI-1/LRP1 axis on the recruitment of MCs in glioma. The connection between high-grade glioma and MC infiltration could contribute to patient tailored therapy and improve patient stratification in future therapeutic trials.

  9. PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1): A KEY FACTOR LINKING FIBRINOLYSIS AND AGE-RELATED SUBCLINICAL AND CLINICAL CONDITIONS

    PubMed Central

    Cesari, Matteo; Pahor, Marco; Incalzi, Raffaele Antonelli

    2010-01-01

    The close relationship existing between aging and thrombosis has growingly been studied in this last decade. The age-related development of a pro-thrombotic imbalance in the fibrinolysis homeostasis has been hypothesized at the basis of this increased cardiovascular and cerebrovascular risk. Fibrinolysis is the resulting of the interactions among multiple plasminogen activators and inhibitors constituing the enzymatic cascade, and ultimately leading to the degradation of fibrin. The plasminogen activator system plays a key role in a wide range of physiological and pathological processes. Plasminogen activator inhibitor-1 (PAI-1) is a member of the superfamily of serine-protease inhibitors (or serpins), and the principal inhibitor of both the tissue-type and the urinary-type plasminogen activator, the two plasminogen activators able to activate plasminogen. In this review, current evidence describing the central role played by PAI-1 in a number of age-related subclinical (i.e., inflammation, atherosclerosis, insulin resistance) and clinical (i.e., obesity, comorbidities, Werner syndrome) conditions is presented. Despite some controversial and unclear issues, PAI-1 represents an extremely promising marker which may become a biological parameter to be growingly considered in the prognostic evaluation, in the disease monitoring, and as treatment target of age-related conditions in the next future. PMID:20626406

  10. Design, synthesis, and SAR of embelin analogues as the inhibitors of PAI-1 (plasminogen activator inhibitor-1).

    PubMed

    Chen, Fanglei; Zhang, Guiping; Hong, Zebin; Lin, Zhonghui; Lei, Min; Huang, Mingdong; Hu, Lihong

    2014-05-15

    The natural product embelin was found to have PAI-1 inhibitory activity with the IC50 value of 4.94μM. Based on the structure of embelin, a series of analogues were designed, synthesized, and evaluated for their ability to inhibit PAI-1. The SAR study on these compounds disclosed that the inhibitory potency largely depended on the hydroxyl groups at C2 and C5, and the length of the alkyl chains at C3 and C6. Compound 11 displayed the best PAI-1 inhibitory potency with the IC50 value of 0.18μM.

  11. Evaluation of 12-lipoxygenase (12-LOX) and plasminogen activator inhibitor 1 (PAI-1) as prognostic markers in prostate cancer.

    PubMed

    Gondek, Tomasz; Szajewski, Mariusz; Szefel, Jarosław; Aleksandrowicz-Wrona, Ewa; Skrzypczak-Jankun, Ewa; Jankun, Jerzy; Lysiak-Szydlowska, Wieslawa

    2014-01-01

    In carcinoma of prostate, a causative role of platelet 12-lipoxygenase (12-LOX) and plasminogen activator inhibitor 1 (PAI-1) for tumor progression has been firmly established in tumor and/or adjacent tissue. Our goal was to investigate if 12-LOX and/or PAI-1 in patient's plasma could be used to predict outcome of the disease. The study comprised 149 patients (age 70±9) divided into two groups: a study group with carcinoma confirmed by positive biopsy of prostate (n=116) and a reference group (n=33) with benign prostatic hyperplasia (BPH). The following parameters were determined by the laboratory test in plasma or platelet-rich plasma: protein level of 12-LOX, PAI-1, thromboglobulin (TGB), prostate specific antigen (PSA), C-reactive protein (CRP), hemoglobin (HGB, and hematocrit (HCT), as well as red (RBC) and white blood cells (WBC), number of platelets (PLT), international normalized ratio of blood clotting (INR), and activated partial thromboplastin time (APTT). The only difference of significance was noticed in the concentration of 12-LOX in platelet rich plasma, which was lower in cancer than in BPH group. Standardization to TGB and platelet count increases the sensitivity of the test that might be used as a biomarker to assess risk for prostate cancer in periodically monitored patients.

  12. Plasminogen activator inhibitor-1 (PAI-1) promoter polymorphism and coronary artery disease in non-insulin-dependent diabetes.

    PubMed

    Mansfield, M W; Stickland, M H; Grant, P J

    1995-10-01

    Elevated levels of PAI-1 are found in coronary artery disease (CAD) and non-insulin-dependent diabetes (NIDDM). PAI-1 may be involved in the pathogenesis of CAD through suppression of fibrinolysis, alternatively the high levels may result from vascular damage. There is evidence that PAI-1 levels are related to genotype at a PAI-1 promoter polymorphism. Genotype at this 4G/5G polymorphism was determined in 160 NIDDM (90 males and 70 females) patients with (n = 38) or without (n = 122) clinical evidence of CAD. Levels of cholesterol were higher (6.5 vs 5.9 mM, p < 0.01) and PAI-1 tended to be higher (PAI-1 activity 23.0 vs 20.4 U/ml) with CAD. The frequency of the 4G/4G genotype was increased and the 5G/5G genotype decreased, in the group CAD compared to those without (p < 0.05). These results suggest that possession of the 4G/4G PAI-1 promoter genotype is a risk factor for the development of CAD in subjects with NIDDM.

  13. Correlation between plasminogen activator inhibitor-1 (PAI-1) promoter 4G/5G polymorphism and metabolic/proinflammatory factors in polycystic ovary syndrome.

    PubMed

    Sales, M F; Sóter, M O; Candido, A L; Fernandes, A P; Oliveira, F R; Ferreira, A C S; Sousa, M O; Ferreira, C N; Gomes, K B

    2013-10-01

    Polycystic Ovary Syndrome (PCOS) is the most common cause of subfertility associated to metabolic disorders. The aim of this study was to correlate metabolic and proinflammatory factors in women with PCOS. The frequency of Plasminogen Activator Inhibitor-1 (PAI-1) promoter 4 G/5 G polymorphism was also compared to healthy controls. We evaluated 79 PCOS and 79 healthy women. PAI-1 levels are positively correlated with proinflammatory factors in PCOS group. 4 G allele in PAI-1 gene was more frequent in PCOS and the 4G/4 G genotype was associated with increased PAI-1 levels. A correlation between insulin resistance and proinflammatory and overweight was also observed. C-reactive protein, serum levels of vascular cell adhesion molecule-1 (sVCAM-1), Lipid Accumulation Product (LAP) and vitamin D are good tools to evaluated factors associated to cardiovascular risk in women with PCOS.

  14. Signalling networks associated with urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 in breast cancer tissues: new insights from protein microarray analysis.

    PubMed

    Wolff, Claudia; Malinowsky, Katharina; Berg, Daniela; Schragner, Kerstin; Schuster, Tibor; Walch, Axel; Bronger, Holger; Höfler, Heinz; Becker, Karl-Friedrich

    2011-01-01

    The urokinase-type plasminogen activator (uPA) and the main uPA inhibitor PAI-1 play important roles in cell migration and invasion in both physiological and pathological contexts. Both factors are clinically applicable predictive markers in node-negative breast cancer patients that are used to stratify patients for adjuvant chemotherapy. In addition to their classical functions in plasmin regulation, both factors are key components in cancer-related cell signalling. Such signalling cascades are well described in cell culture systems, but a better understanding of uPA- and PAI-1-associated signalling networks in clinical tissues is needed. We examined the expression of uPA, PAI-1, and 21 signalling molecules in 201 primary breast cancer tissues using protein microarrays. Expression of uPA was significantly correlated with the expression of ERK and Stat3, while expression of PAI-1 was correlated with the uPA receptor and Akt activation, presumably via integrin and HER-receptor signalling. Analysis of uPA expression did not reveal any significant correlation with staging, grading or age of the patients. The PAI-1 expression was correlated with nodal stage. Network monitoring for uPA and PAI-1 in breast cancer reveals interactions with main signalling cascades and extends the findings from cell culture experiments. Our results reveal possible mechanisms underlying cancer development.

  15. Human circadian system causes a morning peak in prothrombotic plasminogen activator inhibitor-1 (PAI-1) independent of the sleep/wake cycle.

    PubMed

    Scheer, Frank A J L; Shea, Steven A

    2014-01-23

    Serious adverse cardiovascular events peak in the morning, possibly related to increased thrombosis in critical vessels. Plasminogen activator inhibitor-1 (PAI-1), which inhibits fibrinolysis, is a key circulating prothrombotic factor that rises in the morning in humans. We tested whether this morning peak in PAI-1 is caused by the internal circadian system or by behaviors that typically occur in the morning, such as altered posture and physical activity. Twelve healthy adults underwent a 2-week protocol that enabled the distinction of endogenous circadian effects from behavioral and environmental effects. The results demonstrated a robust circadian rhythm in circulating PAI-1 with a peak corresponding to ∼6:30 am. This rhythm in PAI-1 was 8-times larger than changes in PAI-1 induced by standardized behavioral stressors, including head-up tilt and 15-minute cycle exercise. If this large endogenous morning peak in PAI-1 persists in vulnerable individuals, it could help explain the morning peak in adverse cardiovascular events.

  16. Prognostic value of tissue-type plasminogen activator (tPA) and its complex with the type-1 inhibitor (PAI-1) in breast cancer

    PubMed Central

    Witte, J H de; Sweep, C G J; Klijn, J G M; Grebenschikov, N; Peters, H A; Look, M P; Tienoven, ThH van; Heuvel, J J T M; Vries, J Bolt-De; Benraad, ThJ; Foekens, J A

    1999-01-01

    The prognostic value of tissue-type plasminogen activator (tPA) measured in samples derived from 865 patients with primary breast cancer using a recently developed enzyme-linked immunosorbent assay (ELISA) was evaluated. Since the assay could easily be adapted to the assessment of the complex of tPA with its type-1 inhibitor (PAI-1), it was investigated whether the tPA:PAI-1 complex also provides prognostic information. To this end, cytosolic extracts and corresponding detergent extracts of 100 000 g pellets obtained after ultracentrifugation when preparing the cytosolic fractions for routine steroid hormone receptor determination were assayed. Statistically significant correlations were found between the cytosolic levels and those determined in the pellet extracts (Spearman correlation coefficient rs = 0.75, P < 0.001 for tPA and r = 0.50, P < 0.001 for tPA:PAI-1 complex). In both Cox univariate and multivariate analysis elevated levels of (total) tPA determined in the pellet extracts, but not in cytosols, were associated with prolonged relapse-free (RFS) and overall survival (OS). In contrast, high levels of the tPA:PAI-1 complex measured in cytosols, but not in the pellet extracts, were associated with a poor RFS and OS. The prognostic information provided by the cytosolic tPA:PAI-1 complex was comparable to that provided by cytosolic (total) PAI-1. Furthermore, the estimated levels of free, uncomplexed tPA and PAI-1, in cytosols and in pellet extracts, were related to patient prognosis in a similar way as the (total) levels of tPA and PAI-1 respectively. Determination of specific forms of components of the plasminogen activation system, i.e. tPA:PAI-1 complex and free, uncomplexed tPA and/or PAI-1, may be considered a useful adjunct to the analyses of the separate components (tPA and/or PAI-1) and provide valuable additional prognostic information with respect to survival of breast cancer patients. © 1999 Cancer Research Campaign PMID:10390010

  17. PULMONARY LOCALIZATION AND EXPRESSION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) IN HEALTHY OR HYPERTENSIVE RATS EXPOSED TO PARTICULATE MATTER (PM)

    EPA Science Inventory

    PULMONARY LOCALIZATION AND EXPRESSION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1) IN HEALTHY OR HYPERTENSIVE RATS EXPOSED TO PARTICULATE MATTER (PM). GS Backus1, R Vincent2, UP Kodavanti2, 1Curriculum in Toxicology, UNC, Chapel Hill; 2NHEERL, ORD, US EPA, Research Triangle Park,...

  18. Prognostic impact of urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1) in cytosols and pellet extracts derived from 892 breast cancer patients

    PubMed Central

    de Witte, J H; Sweep, C G J; Klijn, J G M; Grebenschikov, N; Peters, H A; Look, M P; van Tienoven, Th H; Heuvel, J J T M; van Putten, W L J; Benraad, Th J; Foekens, J A

    1999-01-01

    To evaluate the clinical relevance of urokinase-type plasminogen activator (uPA) and its type-1 inhibitor (PAI-1) measured by a recently developed enzyme-linked immunosorbent assay (ELISA), we analysed both components in samples derived from 892 patients with primary breast cancer (median follow-up 99 months). The assays were performed in cytosolic extracts as well as in corresponding detergent extracts of pellets obtained after ultracentrifugation, which was carried out when preparing the cytosolic fractions for routine steroid hormone receptor determination. Statistically significant correlations were found between the cytosolic levels and those determined in the pellet extracts (Spearman correlation coefficient rs = 0.60, P < 0.0001 for uPA and rs = 0.65, P < 0.0001 for PAI-1). Furthermore, strong correlations were found between the levels of both uPA (rs = 0.85, P < 0.0001) and PAI-1 (rs = 0.90, P < 0.0001) in the cytosols and their levels previously measured with ELISAs based on commercial reagents. In both Cox univariate and multivariate analysis, high cytosolic levels of uPA or PAI-1 were significantly associated with increased rates of relapse and death. The levels of uPA and PAI-1 in the pellet extracts also provided prognostic information, although to a lesser extent compared with the cytosolic extracts. The prediction of prognosis on the basis of uPA and PAI-1 assessed by an alternative ELISA once again emphasizes the established prognostic role and usefulness of these parameters in selection of breast cancer patients at high or low risk of recurrence. © 1999 Cancer Research Campaign PMID:10098758

  19. A small molecule PAI-1 functional inhibitor attenuates neointimal hyperplasia and vascular smooth muscle cell survival by promoting PAI-1 cleavage.

    PubMed

    Simone, Tessa M; Higgins, Stephen P; Archambeault, Jaclyn; Higgins, Craig E; Ginnan, Roman G; Singer, Harold; Higgins, Paul J

    2015-05-01

    Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of urokinase-and tissue-type plasminogen activators (uPA and tPA), is an injury-response gene implicated in the development of tissue fibrosis and cardiovascular disease. PAI-1 mRNA and protein levels were elevated in the balloon catheter-injured carotid and in the vascular smooth muscle cell (VSMC)-enriched neointima of ligated arteries. PAI-1/uPA complex formation and PAI-1 antiproteolytic activity can be inhibited, via proteolytic cleavage, by the small molecule antagonist tiplaxtinin which effectively increased the VSMC apoptotic index in vitro and attenuated carotid artery neointimal formation in vivo. In contrast to the active full-length serine protease inhibitor (SERPIN), elastase-cleaved PAI-1 (similar to tiplaxtinin) also promoted VSMC apoptosis in vitro and similarly reduced neointimal formation in vivo. The mechanism through which cleaved PAI-1 (CL-PAI-1) stimulates apoptosis appears to involve the TNF-α family member TWEAK (TNF-α weak inducer of apoptosis) and it's cognate receptor, fibroblast growth factor (FGF)-inducible 14 (FN14). CL-PAI-1 sensitizes cells to TWEAK-stimulated apoptosis while full-length PAI-1 did not, presumably due to its ability to down-regulate FN14 in a low density lipoprotein receptor-related protein 1 (LRP1)-dependent mechanism. It appears that prolonged exposure of VSMCs to CL-PAI-1 induces apoptosis by augmenting TWEAK/FN14 pro-apoptotic signaling. This work identifies a critical, anti-stenotic, role for a functionally-inactive (at least with regard to its protease inhibitory function) cleaved SERPIN. Therapies that promote the conversion of full-length to cleaved PAI-1 may have translational implications.

  20. The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues.

    PubMed

    Gettins, Peter G W; Dolmer, Klavs

    2016-01-01

    Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13-800-fold increase in Kd values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association.

  1. The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues.

    PubMed

    Gettins, Peter G W; Dolmer, Klavs

    2016-01-01

    Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13-800-fold increase in Kd values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association. PMID:26555266

  2. Urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) are potential predictive biomarkers in early stage oral squamous cell carcinomas (OSCC).

    PubMed

    Magnussen, Synnøve; Rikardsen, Oddveig G; Hadler-Olsen, Elin; Uhlin-Hansen, Lars; Steigen, Sonja E; Svineng, Gunbjørg

    2014-01-01

    Oral squamous cell carcinoma (OSCC) is often associated with metastatic disease and a poor 5 year survival rate. Patients diagnosed with small tumours generally have a more favourable outcome, but some of these small tumours are aggressive and lead to early death. To avoid harmful overtreatment of patients with favourable prognosis, there is a need for predictive biomarkers that can be used for treatment stratification. In this study we assessed the possibility to use components of the plasminogen activator (PA) system as prognostic markers for OSCC outcome and compared this to the commonly used biomarker Ki-67. A tissue-micro-array (TMA) based immunohistochemical analysis of primary tumour tissue obtained from a North Norwegian cohort of 115 patients diagnosed with OSCC was conducted. The expression of the biomarkers was compared with clinicopathological variables and disease specific death. The statistical analyses revealed that low expression of uPAR (p = 0.031) and PAI-1 (p = 0.021) in the tumour cells was significantly associated with low disease specific death in patients with small tumours and no lymph node metastasis (T1N0). The commonly used biomarker, Ki-67, was not associated with disease specific death in any of the groups of patients analysed. The conclusion is that uPAR and PAI-1 are potential predictive biomarkers in early stage tumours and that this warrants further studies on a larger cohort of patients.

  3. Hesperetin and its sulfate and glucuronide metabolites inhibit TNF-α induced human aortic endothelial cell migration and decrease plasminogen activator inhibitor-1 (PAI-1) levels.

    PubMed

    Giménez-Bastida, Juan Antonio; González-Sarrías, Antonio; Vallejo, Fernando; Espín, Juan Carlos; Tomás-Barberán, Francisco A

    2016-01-01

    Epidemiological, clinical and preclinical studies have reported the protection offered by citrus consumption, mainly orange, against cardiovascular diseases, which is primarily mediated by the antiatherogenic and vasculoprotective effects of the flavanone hesperetin-7-O-rutinoside (hesperidin). However, flavanone aglycones or glycosides are not present in the bloodstream but their derived phase-II metabolites could be the actual bioactive molecules. To date, only a few studies have explored the effects of circulating hesperetin-derived metabolites (glucuronides and sulfates) on endothelial cells. Herein, we describe for the first time the effects of hesperetin 3'-O-glucuronide, hesperetin 7-O-glucuronide, hesperetin 3'-O-sulfate, hesperetin 7-O-sulfate and hesperetin on human aortic endothelial cell (HAEC) migration upon pro-inflammatory stimuli as an essential step to angiogenesis. Hesperetin and its derived metabolites, at physiologically relevant concentrations (1-10 μM), significantly attenuated cell migration in the presence of the pro-inflammatory cytokine TNF-α (50 ng mL(-1)), which was accompanied and perhaps mediated by a significant decrease in the levels of the thrombogenic plasminogen activator inhibitor-1 (PAI-1). However, hesperetin metabolites did not counteract the TNF-α-induced production of pro-inflammatory interleukin-6 (IL-6) and IL-8. We also study here for the first time, the metabolism of hesperetin and its derived metabolites by HAEC with and without a pro-inflammatory stimulus. All these results reinforce the concept according to which circulating phase-II hesperetin metabolites are critical molecules contributing to the cardioprotective effects upon consumption of citrus fruits such as orange.

  4. The effect of plasminogen activator inhibitor-1 -675 4G/5G polymorphism on PAI-1 gene expression and adipocyte differentiation.

    PubMed

    Ozel Demiralp, Duygu; Aktas, Huseyin; Akar, Nejat

    2008-10-01

    Obesity is a complex, multifactorial chronic disease frequently associated with cardiovascular risks, hypertriglyceridemia, low high-density lipoprotein-cholesterol, high blood pressure, and the insulin resistance that appears to be central to the pathogenesis of Type II diabetes. Plasminogen activator inhibitor-1 expression induced in differentiating adipose tissue, but its role in adipogenesis and obesity is poorly understood. Circulating plasminogen activator inhibitor-1 levels are elevated at an early stage of impaired glucose tolerance, resulting in diabetes and metabolic syndrome. Plasminogen activator inhibitor-1 levels are also significantly elevated in the plasma of obese individuals and in adipose tissues of obese mice and humans. Some investigators proposed that the -675 4G/5G polymorphism in plasminogen activator inhibitor-1 promoter caused overexpression of this gene and predisposed carriers to obesity. In this study, we investigated the role of -675 4G/5G polymorphism in plasminogen activator inhibitor-1 promoter in the expression of this gene and the contribution of plasminogen activator inhibitor-1 to adipogenesis. Using a dual-luciferase promoter assay, we determined that the -675 4G/5G polymorphism contributes significantly to overexpression of plasminogen activator inhibitor-1 in the course of adipogenesis. The antidiabetic agents troglitazone and ciglitazone inhibited reporter gene expression driven by wild-type and -675 4G/5G mutant promoter, as well as the expression of endogenous plasminogen activator inhibitor-1, indicating that suppression of plasminogen activator inhibitor-1 expression may contribute to antidiabetic effects of these agents. The results indicate that absence of plasminogen activator inhibitor-1 in adipocytes may protect the cells against insulin resistance by promoting glucose uptake and adipocyte differentiation via a decrease in the peroxisome proliferator activated receptor-gamma expression that modulates the adipocyte

  5. Procedures for the quantitative protein determination of urokinase and its inhibitor, PAI-1, in human breast cancer tissue extracts by ELISA.

    PubMed

    Schmitt, Manfred; Sturmheit, Alexandra S; Welk, Anita; Schnelldorfer, Christel; Harbeck, Nadia

    2006-01-01

    The determination of the protein content of urokinase-type plasminogen activator (uPA) and its inhibitor, PAI-1, in breast cancer tissue extracts is used clinically to identify patients at risk to experience disease recurrence (metastasis) or early death. The serine protease uPA, in concert with its inhibitor PAI-1, promotes tumor cell adhesion, migration, and proliferation, as well as extracellular matrix degradation and, thus, facilitates tumor cell invasion and metastasis. The various technical steps to recover uPA and PAI-1 protein from archived breast cancer tissues and to quantitatively determine uPA and PAI-1 protein content in tumor tissue extracts by enzyme-linked immunosorbent assay (ELISA) are described in detail. The technical steps involved require fresh-frozen breast cancer tissue, a dismembrator machine (ball mill) to pulverize the tissue in the frozen state, detergent (Triton X-100) containing Tris-buffered saline to extract uPA and PAI-1 from the pulverized breast cancer tissue, an ultracentrifuge to separate the detergent fraction from cellular debris, uPA and PAI-1 ELISA kits, protein determination reagents, and a 96-well spectrophotometer (ELISA reader) to assess uPA, PAI-1, and total protein in the detergent extract. The uPA/PAI-1 ELISAs and the protein determination format described are robust and highly sensitive. In addition to the macromethod of tissue disintegration, we present a simple but sensitive micro-extraction procedure using cryostat sections or core biopsies as the source of breast cancer tissue. Such a technique allows rapid and quantitative determination of uPA and PAI-1, even in small breast cancer specimens.

  6. TGF-β induces p53/Smads complex formation in the PAI-1 promoter to activate transcription

    PubMed Central

    Kawarada, Yuki; Inoue, Yasumichi; Kawasaki, Fumihiro; Fukuura, Keishi; Sato, Koichi; Tanaka, Takahito; Itoh, Yuka; Hayashi, Hidetoshi

    2016-01-01

    Transforming growth factor β (TGF-β) signaling facilitates tumor development during the advanced stages of tumorigenesis, but induces cell-cycle arrest for tumor suppression during the early stages. However, the mechanism of functional switching of TGF-β is still unknown, and it is unclear whether inhibition of TGF-β signaling results amelioration or exacerbation of cancers. Here we show that the tumor suppressor p53 cooperates with Smad proteins, which are TGF-β signal transducers, to selectively activate plasminogen activator inhibitor type-1 (PAI-1) transcription. p53 forms a complex with Smad2/3 in the PAI-1 promoter to recruit histone acetyltransferase CREB-binding protein (CBP) and enhance histone H3 acetylation, resulting in transcriptional activation of the PAI-1 gene. Importantly, p53 is required for TGF-β-induced cytostasis and PAI-1 is involved in the cytostatic activity of TGF-β in several cell lines. Our results suggest that p53 enhances TGF-β-induced cytostatic effects by activating PAI-1 transcription, and the functional switching of TGF-β is partially caused by p53 mutation or p53 inactivation during cancer progression. It is expected that these findings will contribute to optimization of TGF-β-targeting therapies for cancer. PMID:27759037

  7. PPAR{alpha} deficiency augments a ketogenic diet-induced circadian PAI-1 expression possibly through PPAR{gamma} activation in the liver

    SciTech Connect

    Oishi, Katsutaka; Uchida, Daisuke; Ohkura, Naoki; Horie, Shuichi

    2010-10-15

    Research highlights: {yields} PPAR{alpha} deficiency augments a ketogenic diet-induced circadian PAI-1 expression. {yields} Hepatic expressions of PPAR{gamma} and PCG-1{alpha} are induced by a ketogenic diet. {yields} PPAR{gamma} antagonist attenuates a ketogenic diet-induced PAI-1 expression. {yields} Ketogenic diet advances the phase of circadian clock in a PPAR{alpha}-independent manner. -- Abstract: An increased level of plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular diseases, and PAI-1 gene expression is under the control of molecular circadian clocks in mammals. We recently showed that PAI-1 expression is augmented in a phase-advanced circadian manner in mice fed with a ketogenic diet (KD). To determine whether peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) is involved in hypofibrinolytic status induced by a KD, we examined the expression profiles of PAI-1 and circadian clock genes in PPAR{alpha}-null KD mice. Chronic administration of bezafibrate induced the PAI-1 gene expression in a PPAR{alpha}-dependent manner. Feeding with a KD augmented the circadian expression of PAI-1 mRNA in the hearts and livers of wild-type (WT) mice as previously described. The KD-induced mRNA expression of typical PPAR{alpha} target genes such as Cyp4A10 and FGF21 was damped in PPAR{alpha}-null mice. However, plasma PAI-1 concentrations were significantly more elevated in PPAR{alpha}-null KD mice in accordance with hepatic mRNA levels. These observations suggest that PPAR{alpha} activation is dispensable for KD-induced PAI-1 expression. We also found that hyperlipidemia, fatty liver, and the hepatic expressions of PPAR{gamma} and its coactivator PCG-1{alpha} were more effectively induced in PPAR{alpha}-null, than in WT mice on a KD. Furthermore, KD-induced hepatic PAI-1 expression was significantly suppressed by supplementation with bisphenol A diglycidyl ether, a PPAR{gamma} antagonist, in both WT and PPAR

  8. PAI-1 and kidney fibrosis

    PubMed Central

    Ma, Li-Jun; Fogo, Agnes B.

    2016-01-01

    Substantial evidence demonstrates a link of increased plasminogen activator inhibitor-1 (PAI-1) and glomerulosclerosis and kidney fibrosis, providing a novel therapeutic option for prevention and treatment of chronic kidney diseases. Several mechanisms contributing to increased PAI-1 will be addressed, including classic key profibrotic factors such as the renin-angiotensin-system (RAS) and transforming growth factor-beta (TGF-β), and novel molecules identified by proteomic analysis, such as thymosin- β4. The fibrotic sequelae caused by increased PAI-1 in kidney depend not only on its classic inhibition of tissue-type and urokinase-type plasminogen activators (tPA and uPA), but also its influence on cell migration. PMID:19273183

  9. Association between PAI-1 polymorphisms and plasma PAI-1 level with sepsis in severely burned patients.

    PubMed

    Chi, Y F; Chai, J K; Yu, Y M; Luo, H M; Zhang, Q X; Feng, R

    2015-01-01

    We investigated the association between plasminogen activator inhibitor-1 (PAI-1) polymorphisms and plasma PAI-1 level with sepsis in severely burned patients. A total of 182 patients with burn areas lager than 30% of the body surface area were enrolled in this study. Peripheral blood samples were obtained from 103 patients with sepsis (sepsis group) and 79 patients without sepsis (control group). An allele-specific polymerase chain reaction assay was used to determine PAI-1 polymorphism 4G/5G distribution. Plasma PAI-1 levels were detected using an enzyme-linked immunosorbent assay. The frequency of the 4G/4G genotype and the 4G allele frequency in the sepsis group were 42.7 and 62.1% respectively, which were significantly higher than those in the control group (P < 0.05). Sepsis patients had a significantly higher plasma PAI-1 level than the control group (P < 0.05). Compared with the 5G/5G genotype, PAI-1 concentrations were significantly higher in the 4G/4G genotype (P < 0.05). The study indicates that the 4G/5G promoter polymorphism of PAI-1 gene may be related to the susceptibility to burn sepsis and that the 4G/4G genotype may be an important genetic risk factor of burn sepsis. Additionally, PAI-1 concentrations in the serum are increased in patients with burn sepsis.

  10. In black South Africans from rural and urban communities, the 4G/5G PAI-1 polymorphism influences PAI-1 activity, but not plasma clot lysis time.

    PubMed

    de Lange, Zelda; Rijken, Dingeman C; Hoekstra, Tiny; Conradie, Karin R; Jerling, Johann C; Pieters, Marlien

    2013-01-01

    Data on genetic and environmental factors influencing PAI-1 levels and their consequent effect on clot lysis in black African populations are limited. We identified polymorphisms in the promoter area of the PAI-1 gene and determined their influence on PAI-1act levels and plasma clot lysis time (CLT). We also describe gene-environment interactions and the effect of urbanisation. Data from 2010 apparently healthy urban and rural black participants from the South African arm of the PURE study were cross-sectionally analysed. The 5G allele frequency of the 4G/5G polymorphism was 0.85. PAI-1act increased across genotypes in the urban subgroup (p = 0.009) but not significantly in the rural subgroup, while CLT did not differ across genotypes. Significant interaction terms were found between the 4G/5G polymorphism and BMI, waist circumference and triglycerides in determining PAI-1act, and between the 4G/5G polymorphism and fibrinogen and fibrinogen gamma prime in determining CLT. The C428T and G429A polymorphisms did not show direct relationships with PAI-1act or CLT but they did influence the association of other environmental factors with PAI-1act and CLT. Several of these interactions differed significantly between rural and urban subgroups, particularly in individuals harbouring the mutant alleles. In conclusion, although the 4G/5G polymorphism significantly affected PAI-1act, it contributed less than 1% to the PAI-1act variance. (Central) obesity was the biggest contributor to PAI-1act variance (12.5%). Urbanisation significantly influenced the effect of the 4G/5G polymorphism on PAI-1act as well as gene-environment interactions for the C428T and G429A genotypes in determining PAI-1act and CLT. PMID:24386152

  11. In Black South Africans from Rural and Urban Communities, the 4G/5G PAI-1 Polymorphism Influences PAI-1 Activity, but Not Plasma Clot Lysis Time

    PubMed Central

    de Lange, Zelda; Rijken, Dingeman C.; Hoekstra, Tiny; Conradie, Karin R.; Jerling, Johann C.; Pieters, Marlien

    2013-01-01

    Data on genetic and environmental factors influencing PAI-1 levels and their consequent effect on clot lysis in black African populations are limited. We identified polymorphisms in the promoter area of the PAI-1 gene and determined their influence on PAI-1act levels and plasma clot lysis time (CLT). We also describe gene-environment interactions and the effect of urbanisation. Data from 2010 apparently healthy urban and rural black participants from the South African arm of the PURE study were cross-sectionally analysed. The 5G allele frequency of the 4G/5G polymorphism was 0.85. PAI-1act increased across genotypes in the urban subgroup (p = 0.009) but not significantly in the rural subgroup, while CLT did not differ across genotypes. Significant interaction terms were found between the 4G/5G polymorphism and BMI, waist circumference and triglycerides in determining PAI-1act, and between the 4G/5G polymorphism and fibrinogen and fibrinogen gamma prime in determining CLT. The C428T and G429A polymorphisms did not show direct relationships with PAI-1act or CLT but they did influence the association of other environmental factors with PAI-1act and CLT. Several of these interactions differed significantly between rural and urban subgroups, particularly in individuals harbouring the mutant alleles. In conclusion, although the 4G/5G polymorphism significantly affected PAI-1act, it contributed less than 1% to the PAI-1act variance. (Central) obesity was the biggest contributor to PAI-1act variance (12.5%). Urbanisation significantly influenced the effect of the 4G/5G polymorphism on PAI-1act as well as gene-environment interactions for the C428T and G429A genotypes in determining PAI-1act and CLT. PMID:24386152

  12. Intracellular Expression of PAI-1 Specific Aptamers Alters Breast Cancer Cell Migration, Invasion and Angiogenesis

    PubMed Central

    Fortenberry, Yolanda M.; Brandal, Stephanie M.; Carpentier, Gilles; Hemani, Malvi; Pathak, Arvind P.

    2016-01-01

    Plasminogen activator inhibitor-1 (PAI-1) is elevated in various cancers, where it has been shown to effect cell migration and invasion and angiogenesis. While, PAI-1 is a secreted protein, its intercellular levels are increased in cancer cells. Consequently, intracellular PAI-1 could contribute to cancer progression. While various small molecule inhibitors of PAI-1 are currently being investigated, none specifically target intracellular PAI-1. A class of inhibitors, termed aptamers, has been used effectively in several clinical applications. We previously generated RNA aptamers that target PAI-1 and demonstrated their ability to inhibit extracellular PAI-1. In the current study we explored the effect of these aptamers on intracellular PAI-1. We transiently transfected the PAI-1 specific aptamers into both MDA-MB-231 human breast cancer cells, and human umbilical vein endothelial cells (HUVECs) and studied their effects on cell migration, invasion and angiogenesis. Aptamer expressing MDA-MB-231 cells exhibited a decrease in cell migration and invasion. Additionally, intracellular PAI-1 and urokinase plasminogen activator (uPA) protein levels decreased, while the PAI-1/uPA complex increased. Moreover, a significant decrease in endothelial tube formation in HUVECs transfected with the aptamers was observed. In contrast, conditioned media from aptamer transfected MDA-MB-231 cells displayed a slight pro-angiogenic effect. Collectively, our study shows that expressing functional aptamers inside breast and endothelial cells is feasible and may exhibit therapeutic potential. PMID:27755560

  13. Small molecules inhibitors of plasminogen activator inhibitor-1 - an overview.

    PubMed

    Rouch, Anne; Vanucci-Bacqué, Corinne; Bedos-Belval, Florence; Baltas, Michel

    2015-03-01

    PAI-1, a glycoprotein from the serpin family and the main inhibitor of tPA and uPA, plays an essential role in the regulation of intra and extravascular fibrinolysis by inhibiting the formation of plasmin from plasminogen. PAI-1 is also involved in pathological processes such as thromboembolic diseases, atherosclerosis, fibrosis and cancer. The inhibition of PAI-1 activity by small organic molecules has been observed in vitro and with some in vivo models. Based on these findings, PAI-1 appears as a potential therapeutic target for several pathological conditions. Over the past decades, many efforts have therefore been devoted to developing PAI-1 inhibitors. This article provides an overview of the publishing activity on small organic molecules used as PAI-1 inhibitors. The chemical synthesis of the most potent inhibitors as well as their biological and biochemical evaluations is also presented.

  14. Functional Stability of Plasminogen Activator Inhibitor-1

    PubMed Central

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

    2014-01-01

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

  15. Tissue-specific PAI-1 gene expression and glycosylation pattern in insulin-resistant old rats.

    PubMed

    Serrano, R; Barrenetxe, J; Orbe, J; Rodríguez, J A; Gallardo, N; Martínez, C; Andrés, A; Páramo, J A

    2009-11-01

    Increased levels of plasminogen activator inhibitor-1 (PAI-1) have been associated with obesity, aging, insulin resistance, and type 2 diabetes, conditions that contribute to increased cardiovascular risk. PAI-1 is expressed in a variety of tissues, but the cellular origin of plasma PAI-1 is unknown. To link insulin resistance, aging, and cardiovascular disease, we examined the expression and glycosylation pattern of PAI-1 in liver and white adipose tissue (WAT) from adult (3 mo) and insulin-resistant old (24 mo) Wistar rats. Glycosylated PAI-1 protein was also purified by affinity chromatography from endothelial culture supernatans to analyze its inhibitory activity. We also analyzed the contribution of adipocytes and stromal vascular cells from WAT to PAI-1 levels with aging. Aging caused a significant increase of PAI-1 mRNA (P < 0.001) in WAT that was predominantly due to the adipocytes and not to stroma-vascular cells, while there was no modification in liver from aged rats. Moreover, PAI-1 expression increased during preadipocyte differentiation (P < 0.001). Furthermore, we found a tissue-dependent PAI-1 glycosylation pattern: adipose tissue only expresses the glycosylated PAI-1 form, whereas the liver mainly expresses the nonglycosylated form. Finally, we also found evidences suggesting that the glycosylated PAI-1 form shows higher inhibitory activity than the nonglycosylated. Our data suggest that WAT may be a major source of the elevated plasma levels of PAI-1 in insulin-resistant old rats. Additionally, the high degree of PAI-1 glycosylation and activity, together with the significant increase in visceral fat in old rats, may well contribute to an increased cardiovascular risk associated with insulin-resistant states.

  16. PAI-1 and TNF-α profiles of adipose tissue in obese cardiovascular disease patients

    PubMed Central

    Bilgic Gazioglu, Sema; Akan, Gokce; Atalar, Fatma; Erten, Gaye

    2015-01-01

    Obesity as a leading preventable cause of death worldwide is closely linked to cardiovascular disease (CVD). Plasma plasminogen activator inhibitor (PAI)-1, a potent inhibitor of plasminogen activation and fibrinolysis, is increased in many clinical situations associated with high incidence of CVD. In the obesity-linked elevation of PAI-1, evidence points to TNF-α as an important regulator of PAI-1 expression in adipose tissue. Background: This study aims to evaluate mediastinal PAI-1 and TNF-α mRNA levels in adipose tissues (AT) and compare serum levels in obesity with and without coronary artery disease (CAD). Patients and methods: Obese patients with (n=37) and without CAD (n=20) were included in the study. Results: The serum levels of PAI-1 and TNF-α were significantly higher in obese patients with CAD compared to obese patients without CAD. PAI-1 mRNA expression was significantly increased in mediastinal adipose tissue (MAT) of obese patients with CAD compared to those without CAD, TNF-α mRNA expressions were found to be higher in EAT (epicardial AT), MAT and SAT (subcutaneous AT) of obese patients with CAD. Conclusions: The study demonstrated a close direct relationship between TNF-α and PAI-1. PAI-1 mRNA expression strongly correlated positively with serum TNF-α in MAT, and TNF-α expressions with PAI-1 serum levels. PMID:26884864

  17. Peroxisome proliferator-activated receptor-α-mediated transcription of miR-301a and miR-454 and their host gene SKA2 regulates endothelin-1 and PAI-1 expression in sickle cell disease.

    PubMed

    Gonsalves, Caryn S; Li, Chen; Malik, Punam; Tahara, Stanley M; Kalra, Vijay K

    2015-01-01

    Endothelin-1 (ET-1) and plasminogen activator inhibitor-1 (PAI-1) play important roles in pulmonary hypertension (PH) in sickle cell disease (SCD). Our previous studies show higher levels of placenta growth factor (PlGF) in SCD correlate with increased plasma levels of ET-1, PAI-1, and other physiological markers of PH. PlGF-mediated ET-1 and PAI-1 expression occurs via activation of hypoxia-inducible factor-1α (HIF-1α). However, relatively little is understood regarding post-transcriptional regulation of PlGF-mediated expression of ET-1 and PAI-1. Herein, we show PlGF treatment of endothelial cells reduced levels of miR-301a and miR-454 from basal levels. In addition, both miRNAs targeted the 3'-UTRs of ET-1 and PAI-1 mRNAs. These results were corroborated in the mouse model of SCD [Berkeley sickle mice (BK-SS)] and in SCD subjects. Plasma levels of miR-454 in SCD subjects were significantly lower compared with unaffected controls, which correlated with higher plasma levels of both ET-1 and PAI-1. Moreover, lung tissues from BK-SS mice showed significantly reduced levels of pre-miR-301a and concomitantly higher levels of ET-1 and PAI-1. Furthermore, we show that miR-301a/miR-454 located in the spindle and kinetochore-associated protein-2 (SKA2) transcription unit was co-transcriptionally regulated by both HIF-1α and peroxisome proliferator-activated receptor-α (PPAR-α) as demonstrated by SKA2 promoter mutational analysis and ChIP. Finally we show that fenofibrate, a PPAR-α agonist, increased the expression of miR-301a/miR-454 and SKA2 in human microvascular endothelial cell line (HMEC) cells; the former were responsible for reduced expression of ET-1 and PAI-1. Our studies provide a potential therapeutic approach whereby fenofibrate-induced miR-301a/miR-454 expression can ameliorate PH and lung fibrosis by reduction in ET-1 and PAI-1 levels in SCD.

  18. Resveratrol suppresses PAI-1 gene expression in a human in vitro model of inflamed adipose tissue.

    PubMed

    Zagotta, Ivana; Dimova, Elitsa Y; Funcke, Jan-Bernd; Wabitsch, Martin; Kietzmann, Thomas; Fischer-Posovszky, Pamela

    2013-01-01

    Increased plasminogen activator inhibitor-1 (PAI-1) levels are associated with a number of pathophysiological complications; among them is obesity. Resveratrol was proposed to improve obesity-related health problems, but the effect of resveratrol on PAI-1 gene expression in obesity is not completely understood. In this study, we used SGBS adipocytes and a model of human adipose tissue inflammation to examine the effects of resveratrol on the production of PAI-1. Treatment of SGBS adipocytes with resveratrol reduced PAI-1 mRNA and protein in a time- and concentration-dependent manner. Further experiments showed that obesity-associated inflammatory conditions lead to the upregulation of PAI-1 gene expression which was antagonized by resveratrol. Although signaling via PI3K, Sirt1, AMPK, ROS, and Nrf2 appeared to play a significant role in the modulation of PAI-1 gene expression under noninflammatory conditions, those signaling components were not involved in mediating the resveratrol effects on PAI-1 production under inflammatory conditions. Instead, we demonstrate that the resveratrol effects on PAI-1 induction under inflammatory conditions were mediated via inhibition of the NF κ B pathway. Together, resveratrol can act as NF κ B inhibitor in adipocytes and thus the subsequently reduced PAI-1 expression in inflamed adipose tissue might provide a new insight towards novel treatment options of obesity. PMID:23819014

  19. Abrogation of plasminogen activator inhibitor-1-vitronectin interaction ameliorates acute kidney injury in murine endotoxemia.

    PubMed

    Gupta, Kamlesh K; Donahue, Deborah L; Sandoval-Cooper, Mayra J; Castellino, Francis J; Ploplis, Victoria A

    2015-01-01

    Sepsis-induced acute kidney injury (AKI) contributes to the high mortality and morbidity in patients. Although the pathogenesis of AKI during sepsis is poorly understood, it is well accepted that plasminogen activator inhibitor-1 (PAI-1) and vitronectin (Vn) are involved in AKI. However, the functional cooperation between PAI-1 and Vn in septic AKI has not been completely elucidated. To address this issue, mice were utilized lacking either PAI-1 (PAI-1-/-) or expressing a PAI-1-mutant (PAI-1R101A/Q123K) in which the interaction between PAI-1 and Vn is abrogated, while other functions of PAI-1 are retained. It was found that both PAI-1-/- and PAI-1R101A/Q123K mice are associated with decreased renal dysfunction, apoptosis, inflammation, and ERK activation as compared to wild-type (WT) mice after LPS challenge. Also, PAI-1-/- mice showed attenuated fibrin deposition in the kidneys. Furthermore, a lack of PAI-1 or PAI-1-Vn interaction was found to be associated with an increase in activated Protein C (aPC) in plasma. These results demonstrate that PAI-1, through its interaction with Vn, exerts multiple deleterious mechanisms to induce AKI. Therefore, targeting of the PAI-1-Vn interaction in kidney represents an appealing therapeutic strategy for the treatment of septic AKI by not only altering the fibrinolytic capacity but also regulating PC activity.

  20. Plasminogen activator inhibitor-1 and diabetic nephropathy.

    PubMed

    Lee, Hi Bahl; Ha, Hunjoo

    2005-10-01

    Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) in the kidney. Decreased ECM degradation as well as increased ECM synthesis plays an important role in ECM remodeling that favours tissue fibrosis. Plasminogen activator (PA)/plasmin/PA inhibitor (PAI) system is involved in ECM degradation and PAI-1 plays a critical role in ECM remodeling in the kidney. Normal human kidneys do not express PAI-1 but PAI-1 is overexpressed in pathologic conditions associated with renal fibrosis including diabetic nephropathy. Reactive oxygen species mediate PAI-1 up-regulation in renal cells cultured under high glucose, hypoxia, and TGF-beta1. Recent studies utilizing PAI-1 deficient mice suggest that PAI-1 induce ECM deposition in diabetic kidney through increased ECM synthesis by TGF-beta1 up-regulation as well as through decreased ECM degradation by suppression of plasmin and MMP-2 activity.

  1. Inhibition of plasminogen activator inhibitor-1 is a potential therapeutic strategy in ovarian cancer.

    PubMed

    Mashiko, Satsuki; Kitatani, Kazuyuki; Toyoshima, Masafumi; Ichimura, Atsuhiko; Dan, Takashi; Usui, Toshinori; Ishibashi, Masumi; Shigeta, Shogo; Nagase, Satoru; Miyata, Toshio; Yaegashi, Nobuo

    2015-01-01

    Plasminogen activator inhibitor (PAI)-1 is predictive of poor outcome in several types of cancer. The present study investigated the biological role for PAI-1 in ovarian cancer and potential of targeted pharmacotherapeutics. In patients with ovarian cancer, PAI-1 mRNA expression in tumor tissues was positively correlated with poor prognosis. To determine the role of PAI-1 in cell proliferation in ovarian cancer, the effects of PAI-1 inhibition were examined in PAI-1-expressing ovarian cancer cells. PAI-1 knockdown by small interfering RNA resulted in significant suppression of cell growth accompanied with G2/M cell cycle arrest and intrinsic apoptosis. Similarly, treatment with the small molecule PAI-1 inhibitor TM5275 effectively blocked cell proliferation of ovarian cancer cells that highly express PAI-1. Together these results suggest that PAI-1 promotes cell growth in ovarian cancer. Interestingly, expression of PAI-1 was increased in ovarian clear cell carcinoma compared with that in serous tumors. Our results suggest that PAI-1 inhibition promotes cell cycle arrest and apoptosis in ovarian cancer and that PAI-1 inhibitors potentially represent a novel class of anti-tumor agents.

  2. PERIOD2 is a circadian negative regulator of PAI-1 gene expression in mice.

    PubMed

    Oishi, Katsutaka; Miyazaki, Koyomi; Uchida, Daisuke; Ohkura, Naoki; Wakabayashi, Miyuki; Doi, Ryosuke; Matsuda, Juzo; Ishida, Norio

    2009-04-01

    An increased level of obesity-induced plasma plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular disease. To determine whether the circadian clock component PERIOD2 (PER2) is involved in the regulation of PAI-1 gene expression, we performed transient transfection assays in vitro, and generated transgenic (Tg) mice overexpressing PER2. We then compared PAI-1 expression in Tg and wild-type (WT) mice with or without obesity induced by a high-fat/high-sucrose diet. PER2 suppressed CLOCK:BMAL1- and CLOCK:BMAL2-dependent transactivation of the PAI-1 promoter in vitro. Furthermore, nuclear translocation is dispensable for PER2 to suppress CLOCK:BMAL1-dependent transactivation of the PAI-1 promoter, because functional loss of the nuclear localization domain did not affect either the interaction with BMAL1 or the suppressive role of PER2. The diurnal expression of clock and clock-controlled genes was disrupted in a gene-specific manner, whereas that of PAI-1 mRNA was significantly damped in the hearts of PER2 Tg mice fed with a normal diet. Obesity-induced plasma PAI-1 increase was significantly suppressed in Tg mice in accordance with cardiac PAI-1 mRNA levels, whereas body weight gain and changes in metabolic parameters were identical between WT and Tg mice. Endogenous PAI-1 gene expression induced by transforming growth factor-beta1 was significantly attenuated in embryonic fibroblasts derived from Tg mice compared with those from WT mice. Our results demonstrated that PER2 represses PAI-1 gene transcription in a BMAL1/2-dependent manner. The present findings also suggest that PER2 attenuates obesity-induced hypofibrinolysis by downregulating PAI-1 expression independently of metabolic disorders.

  3. Increased Umbilical Cord PAI-1 Levels in Placental Insufficiency Are Associated with Fetal Hypoxia and Angiogenesis

    PubMed Central

    Seferovic, Maxim D.; Gupta, Madhulika B.

    2016-01-01

    In intrauterine growth restriction (IUGR), a subset of pregnancies undergoes placental vascular dysregulation resulting in restricted blood flow and fetal hypoxemia. Altered transcription of hypoxic regulated plasminogen activator inhibitor 1 (PAI-1) has been associated with pregnancy complications and angiogenic regulation. Here we assessed circulating PAI-1 as an indicator of placental insufficiency. Venous umbilical PAI-1 of hypoxemic (VpO2 20 versus 35 mmHg, p < 0.0001) placental insufficient pregnancies (resistance index 0.9 versus 0.63, p < 0.05) (n = 18) was compared to controls (n = 12). PAI-1 was increased (~10-fold, p < 0.001) and had a positive predictive ratio of 6.7. Further, PAI-1 levels correlated to blood oxygen (r = −0.68, p < 0.0001). The plasma's angiogenic potency measured in vitro was associated with umbilical cord blood PAI-1 levels (r = 0.65, p < 0.01). This association was attenuated by PAI-1 inhibiting antibody (p < 0.001). The results demonstrate PAI-1 as a potential marker of placental insufficiency and identify its close association with pathological hypoxia and angiogenesis in a subset of growth restricted pregnancies. PMID:26903689

  4. PAI-1 Expression Is Required for HDACi-Induced Proliferative Arrest in ras-Transformed Renal Epithelial Cells

    PubMed Central

    Higgins, Stephen P.; Higgins, Craig E.; Higgins, Paul J.

    2011-01-01

    Malignant transformation of mammalian cells with ras family oncogenes results in dramatic changes in cellular architecture and growth traits. The generation of flat revertants of v-K-ras-transformed renal cells by exposure to the histone deacetylase inhibitor sodium butyrate (NaB) was previously found to be dependent on transcriptional activation of the PAI-1 (SERPINE1) gene (encoding the type-1 inhibitor of urokinase and tissue-type plasminogen activators). NaB-initiated PAI-1 expression preceded induced cell spreading and entry into G1 arrest. To assess the relevance of PAI-1 induction to growth arrest in this cell system more critically, two complementary approaches were used. The addition of a stable, long half-life, recombinant PAI-1 mutant to PAI-1-deficient v-K-ras-/c-Ha-ras-transformants or to PAI-1 functionally null, NaB-resistant, 4HH cells (engineered by antisense knockdown of PAI-1 mRNA transcripts) resulted in marked cytostasis in the absence of NaB. The transfection of ras-transformed cells with the Rc/CMVPAI expression construct, moreover, significantly elevated constitutive PAI-1 synthesis (10- to 20-fold) with a concomitant reduction in proliferative rate. These data suggest that high-level PAI-1 expression suppresses growth of chronic ras-oncogene transformed cells and is likely a major cytostatic effector of NaB exposure. PMID:21912547

  5. Male-female differences in the genetic regulation of t-PA and PAI-1 levels in a Ghanaian population.

    PubMed

    Schoenhard, J A; Asselbergs, F W; Poku, K A; Stocki, S A; Gordon, S; Vaughan, D E; Brown, N J; Moore, J H; Williams, Scott M

    2008-12-01

    Tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) directly influence thrombus formation and degradation, and have been identified as risk factors for thromboembolic disease. Prior studies investigated determinants of t-PA and PAI-1 expression, but mainly in Caucasian subjects. The aim of this study was to identify the contributions of genetic and other factors to inter-individual variation in plasma levels of t-PA and PAI-1 in a large-scale population-based sample from urban West Africa. t-PA, PAI-1 and several demographic, anthropometric, and metabolic parameters were measured in 992 residents of Sunyani, the capital of the Brong-Ahafo region of Ghana. In addition, nine gene polymorphisms associated with components of the renin-angiotensin and fibrinolytic systems were determined. We found that BMI, systolic and diastolic blood pressure, total cholesterol, glucose, and triglycerides were all significant predictors of t-PA and PAI-1 in both females and males. In addition, a significant relationship was found between the PAI-1 4G/5G (rs1799768) polymorphism on PAI-1 levels in females, the TPA I/D (rs4646972) polymorphism on t-PA and PAI-1 in males, the renin (rs3730103) polymorphism on t-PA and PAI-1 in males, the ethanolamine kinase 2 (rs1917542) polymorphism on PAI-1 in males, and the renin (rs1464816) polymorphism on t-PA in females and on PAI-1 in males. This study of urban West Africans shows that t-PA and PAI-1 levels are determined by both genetic loci of the fibrinolytic and renin-angiotensin systems and other factors often associated with cardiovascular disease, and that genetic factors differ between males and females.

  6. Ascochlorin suppresses TGF-β1-induced PAI-1 expression through the inhibition of phospho-EGFR in rat kidney fibroblast cells.

    PubMed

    Cho, Hyun-Ji; Kang, Jeong-Han; Jeong, Ji-Hak; Jeong, Yun-Jeong; Park, Kwan-Kyu; Park, Yoon-Yub; Moon, Yong-Suk; Kim, Hong-Tae; Chung, Il-Kyung; Kim, Cheorl-Ho; Chang, Hyeun-Wook; Chang, Young-Chae

    2012-04-01

    Fibrosis is induced by the excessive and abnormal deposition of extracellular matrix (ECM) with various growth factors in tissues. Transforming growth factor-β1 (TGF-β1), the growth factor involved in fibrosis, modulates ECM synthesis and accumulation. TGF-β1 enhances the production of stimulators of ECM synthesis such as plasminogen activator inhibitor type 1 (PAI-1). As such, PAI-1 expression directly influences the proteolysis, invasion, and accumulation of ECM. It was shown in this study that ascochlorin, a prenylpenl antiobiotic, prevents the expression of profibrotic factors, such as PAI-1 and collagen type I, and that the TGF-β1-induced PAI-1 promoter activity is inhibited by ascochlorin. Ascochlorin abolishes the phosphorylation of the EGFR-MEK-ERK signaling pathway to regulate the TGF-β1-induced expression of PAI-1 without the inhibition of TβRII phosphorylation. Furthermore, the MEK inhibitor and EGFR siRNA block PAI-1 expression, and the Raf-1, MEK, and ERK signaling pathways for the regulation of PAI-1 expression. Ascochlorin suppresses the matrix metalloproteinases (MMPs) activity to activate the heparin-binding EGF-like growth factor (HB-EGF), to induce the phosphorylation of EGFR, and the MMPs inhibitor suppresses EGFR phosphorylation and the PAI-1 mRNA levels. These results suggest that ascochlorin prevents the expression of PAI-1 via the inhibition of an EGFR-dependent signal transduction pathway activated by MMPs.

  7. Aldosterone perturbs adiponectin and PAI-1 expression and secretion in 3T3-L1 adipocytes.

    PubMed

    Li, P; Zhang, X-N; Pan, C-M; Sun, F; Zhu, D-L; Song, H-D; Chen, M-D

    2011-06-01

    Aldosterone is considered as a new cardiovascular risk factor that plays an important role in metabolic syndrome; however, the underlying mechanism of these effects is not clear. Hypoadiponectinemia and elevated circulating concentration of plasminogen activator inhibitor-1 (PAI-1) are causally associated with obesity-related insulin resistance and cardiovascular disease. The aim of the present study is to investigate the effect of aldosterone on the production of adiponectin and PAI-1 in 3T3-L1 adipocytes. Northern and Western blot analyses revealed that aldosterone treatment inhibited adiponectin mRNA expression and secretion and simultaneously enhanced PAI-1 mRNA expression and secretion in a time- and dose-dependent manner. Rosiglitazone did not prevent aldosterone's effect on adiponectin or PAI-1 expression. In contrast, tumor necrosis factor (TNF)-α produced dramatic synergistic effects on adiponectin and PAI-1 expression when added together with aldosterone. Furthermore, the effects of aldosterone on adiponectin and PAI-1 expression appear to be mediated through glucocorticoid receptor (GR) but not mineralocorticoid receptor (MR). These results suggest that the effects of aldosterone on adiponectin and PAI-1 production are one of the underlying mechanisms linking it to insulin resistance, metabolic syndrome and cardiovascular disease. PMID:21667402

  8. Plasminogen activator inhibitor type 1 interacts with alpha3 subunit of proteasome and modulates its activity.

    PubMed

    Boncela, Joanna; Przygodzka, Patrycja; Papiewska-Pajak, Izabela; Wyroba, Elzbieta; Osinska, Magdalena; Cierniewski, Czeslaw S

    2011-02-25

    Plasminogen activator inhibitor type-1 (PAI-1), a multifunctional protein, is an important physiological regulator of fibrinolysis, extracellular matrix homeostasis, and cell motility. Recent observations show that PAI-1 may also be implicated in maintaining integrity of cells, especially with respect to cellular proliferation or apoptosis. In the present study we provide evidence that PAI-1 interacts with proteasome and affects its activity. First, by using the yeast two-hybrid system, we found that the α3 subunit of proteasome directly interacts with PAI-1. Then, to ensure that the PAI-1-proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after transfection of HeLa cells with pCMV-PAI-1 and coimmunoprecipitation of both proteins with anti-PAI-1 antibodies. Subsequently, cellular distribution of the PAI-1-proteasome complexes was established by immunogold staining and electron microscopy analyses. Both proteins appeared in a diffuse cytosolic pattern but also could be found in a dense perinuclear and nuclear location. Furthermore, PAI-1 induced formation of aggresomes freely located in endothelial cytoplasm. Increased PAI-1 expression abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-1 and pd2EGFP-N1 and prevented degradation of p53 as well as IκBα, as evidenced both by confocal microscopy and Western immunoblotting.

  9. Plasminogen Activator Inhibitor Type 1 Interacts with α3 Subunit of Proteasome and Modulates Its Activity*

    PubMed Central

    Boncela, Joanna; Przygodzka, Patrycja; Papiewska-Pajak, Izabela; Wyroba, Elzbieta; Osinska, Magdalena; Cierniewski, Czeslaw S.

    2011-01-01

    Plasminogen activator inhibitor type-1 (PAI-1), a multifunctional protein, is an important physiological regulator of fibrinolysis, extracellular matrix homeostasis, and cell motility. Recent observations show that PAI-1 may also be implicated in maintaining integrity of cells, especially with respect to cellular proliferation or apoptosis. In the present study we provide evidence that PAI-1 interacts with proteasome and affects its activity. First, by using the yeast two-hybrid system, we found that the α3 subunit of proteasome directly interacts with PAI-1. Then, to ensure that the PAI-1-proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after transfection of HeLa cells with pCMV-PAI-1 and coimmunoprecipitation of both proteins with anti-PAI-1 antibodies. Subsequently, cellular distribution of the PAI-1-proteasome complexes was established by immunogold staining and electron microscopy analyses. Both proteins appeared in a diffuse cytosolic pattern but also could be found in a dense perinuclear and nuclear location. Furthermore, PAI-1 induced formation of aggresomes freely located in endothelial cytoplasm. Increased PAI-1 expression abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-1 and pd2EGFP-N1 and prevented degradation of p53 as well as IκBα, as evidenced both by confocal microscopy and Western immunoblotting. PMID:21135093

  10. Plasminogen Activator Inhibitor-1 and Vitronectin Expression Level and Stoichiometry Regulate Vascular Smooth Muscle Cell Migration through Physiological Collagen Matrices

    PubMed Central

    Garg, N.; Goyal, N.; Strawn, T. L.; Wu, J.; Mann, K. M.; Lawrence, D. A.; Fay, W. P.

    2010-01-01

    Summary Background Vascular smooth muscle cell (VSMC) migration is a critical process in arterial remodeling. Purified plasminogen activator inhibitor-1 (PAI-1) is reported to both promote and inhibit VSMC migration on 2-dimensional (D) surfaces. Objective To determine the effects of PAI-1 and vitronectin (VN) expressed by VSMC themselves on migration through physiological collagen matrices. Methods We studied migration of wild-type (WT), PAI-1-deficient, VN-deficient, PAI-1/VN doubly-deficient (DKO), and PAI-1-transgenic (Tg) VSMC through 3-D collagen gels. Results WT VSMC migrated significantly slower than PAI-1- and VN-deficient VSMC, but significantly faster than DKO VSMC. Experiments with recombinant PAI-1 suggested that basal VSMC PAI-1 expression inhibits migration by binding VN, which is secreted by VSMC and binds collagen. However, PAI-1-over-expressing Tg VSMC migrated faster than WT VSMC. Reconstitution experiments with recombinant PAI-1 mutants suggested that the pro-migratory effect of PAI-1 over-expression required its anti-plasminogen activator (PA) and LDL receptor-related protein (LRP) binding functions, but not VN binding. While promoting VSMC migration in the absence of PAI-1, VN inhibited the pro-migratory effect of active PAI-1. Conclusions In isolation, VN and PAI-1 are each pro-migratory. However, via formation of a high-affinity, non-motogenic complex, PAI-1 and VN each buffers the other's pro-migratory effect. The level of PAI-1 expression by VSMC and the concentration of VN in extracellular matrix are critical determinants of whether PAI-1 and VN promote or inhibit migration. These findings help to rectify previously conflicting reports and suggest that PAI-1/VN stoichiometry plays an important role in VSMC migration and vascular remodeling. PMID:20492459

  11. Orphan nuclear receptor small heterodimer partner inhibits angiotensin II- stimulated PAI-1 expression in vascular smooth muscle cells.

    PubMed

    Lee, Kyeong-Min; Seo, Hye-Young; Kim, Mi-Kyung; Min, Ae-Kyung; Ryu, Seong-Yeol; Kim, Yoon-Nyun; Park, Young Joo; Choi, Hueng-Sik; Lee, Ki-Up; Park, Wan-Ju; Park, Keun-Gyu; Lee, In-Kyu

    2010-01-31

    Angiotensin II is a major effector molecule in the development of cardiovascular disease. In vascular smooth muscle cells (VSMCs), angiotensin II promotes cellular proliferation and extracellular matrix accumulation through the upregulation of plasminogen activator inhibitor-1 (PAI-1) expression. Previously, we demonstrated that small heterodimer partner (SHP) represses PAI-1 expression in the liver through the inhibition of TGF-beta signaling pathways. Here, we investigated whether SHP inhibited angiotensin II-stimulated PAI-1 expression in VSMCs. Adenovirus-mediated overexpression of SHP (Ad- SHP) in VSMCs inhibited angiotensin II- and TGF-beta-stimulated PAI-1 expression. Ad-SHP also inhibited angiotensin II-, TGF-beta- and Smad3-stimulated PAI-1 promoter activity, and angiotensin II-stimulated AP-1 activity. The level of PAI-1 expression was significantly higher in VSMCs of SHP(-/-) mice than wild type mice. Moreover, loss of SHP increased PAI-1 mRNA expression after angiotensin II treatment. These results suggest that SHP inhibits PAI-1 expression in VSMCs through the suppression of TGF-beta/Smad3 and AP-1 activity. Thus, agents that target the induction of SHP expression in VSMCs might help prevent the development and progression of atherosclerosis.

  12. Plasminogen Activator Inhibitor-1 in Cancer: Rationale and Insight for Future Therapeutic Testing.

    PubMed

    Placencio, Veronica R; DeClerck, Yves A

    2015-08-01

    Despite its function as an inhibitor of urokinase and tissue-type plasminogen activator (PA), PA inhibitor-1 (PAI-1) has a paradoxical protumorigenic role in cancer, promoting angiogenesis and tumor cell survival. In this review, we summarize preclinical evidence in support of the protumorigenic function of PAI-1 that has led to the testing of small-molecule PAI-1 inhibitors, initially developed as antithrombotic agents, in animal models of cancer. The review discusses the challenges and the opportunities that lay ahead to the development of efficacious and nontoxic PAI-1 inhibitors as anticancer agents.

  13. Intra-airway administration of small interfering RNA targeting plasminogen activator inhibitor-1 attenuates allergic asthma in mice.

    PubMed

    Miyamoto, Shintaro; Hattori, Noboru; Senoo, Tadashi; Onari, Yojiro; Iwamoto, Hiroshi; Kanehara, Masashi; Ishikawa, Nobuhisa; Fujitaka, Kazunori; Haruta, Yoshinori; Murai, Hiroshi; Yokoyama, Akihito; Kohno, Nobuoki

    2011-12-01

    Recent studies suggest that plasminogen activator inhibitor-1 (PAI-1), a major inhibitor of the fibrinolytic system, may promote the development of asthma. To further investigate the significance of PAI-1 in the pathogenesis of asthma and determine the possibility that PAI-1 could be a therapeutic target for asthma, this study was conducted. First, PAI-1 levels in induced sputum (IS) from asthmatic subjects and healthy controls were measured. In asthmatic subjects, IS PAI-1 levels were elevated, compared with that of healthy controls, and were significantly higher in patients with long-duration asthma compared with short-duration asthma. PAI-1 levels were also found to correlate with IS transforming growth factor-β levels. Then, acute and chronic asthma models induced by ovalbumin were established in PAI-1-deficient mice and wild-type mice that received intra-airway administrations of small interfering RNA against PAI-1 (PAI-1-siRNA). We could demonstrate that eosinophilic airway inflammation and airway hyperresponsiveness were reduced in an acute asthma model, and airway remodeling was suppressed in a chronic asthma model in both PAI-1-deficient mice and wild-type mice that received intra-airway administration of PAI-1-siRNA. These results indicate that PAI-1 is strongly involved in the pathogenesis of asthma, and intra-airway administration of PAI-1-siRNA may be able to become a new therapeutic approach for asthma.

  14. Plasminogen activator inhibitor-1 in aging.

    PubMed

    Yamamoto, Koji; Takeshita, Kyosuke; Saito, Hidehiko

    2014-09-01

    Plasminogen activator inhibitor-1 (PAI-1), a principal inhibitor of fibrinolysis, is induced in thrombotic, fibrotic, and cardiovascular diseases, which in turn primarily afflict the older population. This induction of PAI-1 may play an important role in the pathology of these diseases as PAI-1 can regulate the dissolution of fibrin and also inhibit the degradation of the extracellular matrix by reducing plasmin generation. PAI-1 expression is elevated in aged individuals and is significantly upregulated in a variety of pathologies associated with the process of aging, including myocardial and cerebral infarction, vascular (athero) sclerosis, cardiac and lung fibrosis, metabolic syndromes (e.g., hypertension, hyperlipidemia, and insulin resistance), cancer, and inflammatory/stress responses. Thus, PAI-1 may play a critical role in the development of aging-associated pathological changes. In addition, PAI-1 is recognized as a marker of senescence and a key member of a group of proteins collectively known as the senescence-messaging secretome. In this review, we highlight the role of PAI-1 in the pathophysiology of aging and aging-associated disorders.

  15. Plasminogen Activator Inhibitor-1 Controls Vascular Integrity by Regulating VE-Cadherin Trafficking

    PubMed Central

    Daniel, Anna E.; Timmerman, Ilse; Kovacevic, Igor; Hordijk, Peter L.; Adriaanse, Luc; Paatero, Ilkka; Belting, Heinz-Georg; van Buul, Jaap D.

    2015-01-01

    Background Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is expressed and secreted by endothelial cells. Patients with PAI-1 deficiency show a mild to moderate bleeding diathesis, which has been exclusively ascribed to the function of PAI-1 in down-regulating fibrinolysis. We tested the hypothesis that PAI-1 function plays a direct role in controlling vascular integrity and permeability by keeping endothelial cell-cell junctions intact. Methodology/Principal Findings We utilized PAI-039, a specific small molecule inhibitor of PAI-1, to investigate the role of PAI-1 in protecting endothelial integrity. In vivo inhibition of PAI-1 resulted in vascular leakage from intersegmental vessels and in the hindbrain of zebrafish embryos. In addition PAI-1 inhibition in human umbilical vein endothelial cell (HUVEC) monolayers leads to a marked decrease of transendothelial resistance and disrupted endothelial junctions. The total level of the endothelial junction regulator VE-cadherin was reduced, whereas surface VE-cadherin expression was unaltered. Moreover, PAI-1 inhibition reduced the shedding of VE-cadherin. Finally, we detected an accumulation of VE-cadherin at the Golgi apparatus. Conclusions/Significance Our findings indicate that PAI-1 function is important for the maintenance of endothelial monolayer and vascular integrity by controlling VE-cadherin trafficking to and from the plasma membrane. Our data further suggest that therapies using PAI-1 antagonists like PAI-039 ought to be used with caution to avoid disruption of the vessel wall. PMID:26714278

  16. New role of plasminogen activator inhibitor-1 in alcohol-induced liver injury

    PubMed Central

    Arteel, Gavin E

    2008-01-01

    Plasminogen activator inhibitor-1 (PAI-1) is the main inhibitor of plasminogen activators, thereby playing a major role in fibrinolysis. Whereas hyperfibrinolysis is common in alcoholic cirrhosis, hypofibrinolysis (driven mostly by elevated levels of PAI-1) is common during the development of alcoholic liver disease (ALD). However, whether or not PAI-1 plays a causal role in the development of ALD has been unclear. The role of PAI-1 was therefore investigated in models of early (steatosis), intermediate (inflammation/necrosis) and late (fibrosis) stages of alcoholic liver disease. For example, hepatic steatosis caused by both acute and chronic ethanol was blunted by inhibiting PAI-1 activation. This effect of inhibiting PAI-1 appears to be mediated, at least in part, by an increase in very low-density lipoprotein (VLDL) synthesis in the absence of PAI-1. The results from that study also indicated that PAI-1 plays a critical role in both acute and chronic hepatic inflammation. Lastly, knocking out PAI-1 potently protected against experimental hepatic fibrosis; the mechanism of this protective effect appears to be mediated predominantly by extracellular matrix (ECM) resolution by matrix metalloproteases, which are indirectly inhibited by PAI-1. In summary, targeting PAI-1 protects against all three stages of ALD in model systems. The mechanisms by which PAI-1 contributes to these disease stages appear to not only involve the ‘classical’ function of PAI-1 (i.e. in mediating fibrinolysis), but also other functions of this protein. These data support a role of PAI-1 in the initiation and progression of ALD, and suggest that PAI-1 may be a useful target for clinical therapy to halt or blunt disease progression. PMID:18336665

  17. Suppression of PAI-1 expression through inhibition of the EGFR-mediated signaling cascade in rat kidney fibroblast by ascofuranone.

    PubMed

    Cho, Hyun-Ji; Kang, Jeong-Han; Kim, Teoan; Park, Kwang-Kyun; Kim, Cheorl-Ho; Lee, In-Seon; Min, Kwan-Sik; Magae, Junji; Nakajima, Hiroo; Bae, Young-Seuk; Chang, Young-Chae

    2009-05-15

    Fibrosis in glomerulosclerosis causes progressive loss of renal function. Transforming growth factor (TGF)-beta, one of the major profibrotic cytokines, induces the synthesis of plasminogen activator inhibitor (PAI)-1, a factor that plays a crucial role in the development of fibrosis. Here, we found that an isoprenoid antibiotic, ascofuranone, suppresses expression of profibrotic factors including matrix proteins and PAI-1 induced by TGF-beta in renal fibroblasts. Ascofuranone selectively inhibits phosphorylation of epidermal growth factor receptor (EGFR), and downstream kinases such as Raf-1, MEK-1/2, and ERK-1/2. PAI-1 transcription also is suppressed by treatment with kinase inhibitors for MEK-1/2 or EGFR, and with small interfering RNA for EGFR. Ascofuranone inhibits cellular metalloproteinase activity, and an inhibitor of metalloproteinases suppresses EGFR phosphorylation and PAI-1 transcription. These results suggest that ascofuranone suppresses expression of profibrotic factors through the inhibition of an EGFR-dependent signal transduction pathway activated by metalloproteinases.

  18. 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.

  19. Construction of a plasminogen activator inhibitor-1 variant without measurable affinity to vitronectin but otherwise normal.

    PubMed

    Jensen, Jan K; Durand, Michelle K V; Skeldal, Sune; Dupont, Daniel M; Bødker, Julie S; Wind, Troels; Andreasen, Peter A

    2004-01-01

    Vitronectin (VN) and plasminogen activator inhibitor-1 (PAI-1) have important functional interactions: VN stabilises the protease inhibitory activity of PAI-1 and PAI-1 inhibits binding of adhesion receptors to VN. Having previously mapped the PAI-1 binding area for VN, we have now constructed a PAI-1 variant, R103A-M112A-Q125A, without measurable affinity to VN, but with full protease inhibitory activity and endocytosis receptor binding. As a tool for evaluating the physiological and pathophysiological functions of the PAI-1-VN interaction, our new variant is far superior to the previously widely used PAI-1 variant Q125K, which we have found possesses an only about 10-fold reduced affinity to VN.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  1. Vitronectin-binding PAI-1 protects against the development of cardiac fibrosis through interaction with fibroblasts.

    PubMed

    Zhong, Jianyong; Yang, Hai-Chun; Kon, Valentina; Fogo, Agnes B; Lawrence, Daniel A; Ma, Ji

    2014-06-01

    Plasminogen activator inhibitor-1 (PAI-1) promotes or abates fibrotic processes occurring in different organs. Binding of PAI-1 to vitronectin, an extracellular matrix component, may inhibit vitronectin-integrin complex-mediated cellular responses in pathophysiological conditions. To investigate the importance of plasmin suppression vs vitronectin-binding pathways of PAI-1 in cardiac fibrosis, we studied uninephrectomized mice fed a high salt diet and infused with angiotensin II (Ang II) together with different PAI-1 variants, including PAI-1AK (AK) that inhibits plasminogen activators but does not bind vitronectin, PAI-1RR (RR) that binds vitronectin but does not have protease inhibitory effects or control PAI-1 (CPAI), the control mutant that has similar molecular backbone and half-life as AK and RR while retaining all functions of native PAI-1. Compared with RR and CPAI, non-vitronectin-binding AK significantly increased expression of cardiac fibroblast marker, periostin (Ang+AK 8.40±3.55 vs Ang+RR 2.23±0.44 and Ang+CPAI 2.33±0.12% positive area, both P<0.05) and cardiac fibrosis (Ang+AK 1.79±0.26% vs Ang+RR 0.91±0.18% and Ang+CPAI 0.81±0.12% fibrotic area, both P<0.05), as well as Col1 mRNA (Ang+AK 12.81±1.84 vs Ang+RR 4.04±1.06 and Ang+CPAI 5.23±1.21 fold increase, both P<0.05). To elucidate mechanisms underlying the protective effects of vitronectin-binding PAI-1 against fibrosis, fibroblasts from normal adult human ventricles were stimulated with Ang and different PAI-1 variants. Protease inhibitory AK and CPAI increased supernatant fibronectin, while decreasing plasminogen activator/plasmin activities and matrix metalloproteinase. RR and CPAI variants significantly reduced fibroblast expression of integrin β3, vitronectin level in the supernatant and fibroblast adhesion to vitronectin compared with the non-vitronectin-binding AK. Further, RR and CPAI preserved apoptotic, decreased anti-apoptotic and proliferative activities in fibroblasts. Thus

  2. Monitoring PAI-1 and VEGF Levels in 6 Human Squamous Cell Carcinoma Xenografts During Fractionated Irradiation

    SciTech Connect

    Bayer, Christine; Kielow, Achim; Schilling, Daniela; Maftei, Constantin-Alin; Zips, Daniel; Yaromina, Ala; Baumann, Michael; Molls, Michael; Multhoff, Gabriele

    2012-11-01

    Purpose: Previous studies have shown that the plasminogen activator inhibitor type-1 (PAI-1) and vascular endothelial growth factor (VEGF) are regulated by hypoxia and irradiation and are involved in neoangiogenesis. The aim of this study was to determine in vivo whether changes in PAI-1 and VEGF during fractionated irradiation could predict for radiation resistance. Methods and Materials: Six xenografted tumor lines from human squamous cell carcinomas (HSCC) of the head and neck were irradiated with 0, 3, 5, 10, and 15 daily fractions of 2 Gy. The PAI-1 and VEGF antigen levels in tumor lysates were determined by enzyme-linked immunosorbent assay kits. The amounts of PAI-1 and VEGF were compared with the dose to cure 50% of tumors (TCD{sub 50}). Colocalization of PAI-1, pimonidazole (hypoxia), CD31 (endothelium), and Hoechst 33342 (perfusion) was examined by immunofluorescence. Results: Human PAI-1 and VEGF (hVEGF) expression levels were induced by fractionated irradiation in UT-SCC-15, UT-SCC-14, and UT-SCC-5 tumors, and mouse VEGF (msVEGF) was induced only in UT-SCC-5 tumors. High hVEGF levels were significantly associated with radiation sensitivity after 5 fractions (P=.021), and high msVEGF levels were significantly associated with radiation resistance after 10 fractions (P=.007). PAI-1 staining was observed in the extracellular matrix, the cytoplasm of fibroblast-like stroma cells, and individual tumor cells at all doses of irradiation. Colocalization studies showed PAI-1 staining close to microvessels. Conclusions: These results indicate that the concentration of tumor-specific and host-specific VEGF during fractionated irradiation could provide considerably divergent information for the outcome of radiation therapy.

  3. Role of plasminogen activator inhibitor-1 in glucocorticoid-induced diabetes and osteopenia in mice.

    PubMed

    Tamura, Yukinori; Kawao, Naoyuki; Yano, Masato; Okada, Kiyotaka; Okumoto, Katsumi; Chiba, Yasutaka; Matsuo, Osamu; Kaji, Hiroshi

    2015-06-01

    Long-term use of glucocorticoids (GCs) causes numerous adverse effects, including glucose/lipid abnormalities, osteoporosis, and muscle wasting. The pathogenic mechanism, however, is not completely understood. In this study, we used plasminogen activator inhibitor-1 (PAI-1)-deficient mice to explore the role of PAI-1 in GC-induced glucose/lipid abnormalities, osteoporosis, and muscle wasting. Corticosterone markedly increased the levels of circulating PAI-1 and the PAI-1 mRNA level in the white adipose tissue of wild-type mice. PAI-1 deficiency significantly reduced insulin resistance and glucose intolerance but not hyperlipidemia induced by GC. An in vitro experiment revealed that active PAI-1 treatment inhibits insulin-induced phosphorylation of Akt and glucose uptake in HepG2 hepatocytes. However, this was not observed in 3T3-L1 adipocytes and C2C12 myotubes, indicating that PAI-1 suppressed insulin signaling in hepatocytes. PAI-1 deficiency attenuated the GC-induced bone loss presumably via inhibition of apoptosis of osteoblasts. Moreover, the PAI-1 deficiency also protected from GC-induced muscle loss. In conclusion, the current study indicated that PAI-1 is involved in GC-induced glucose metabolism abnormality, osteopenia, and muscle wasting in mice. PAI-1 may be a novel therapeutic target to mitigate the adverse effects of GC.

  4. Metals affect the structure and activity of human plasminogen activator inhibitor-1. II. Binding affinity and conformational changes

    PubMed Central

    Thompson, Lawrence C; Goswami, Sumit; Peterson, Cynthia B

    2011-01-01

    Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. The lifespan of the active form of PAI-1 is modulated via interaction with the plasma protein, vitronectin, and various metal ions. These metal ions fall into two categories: Type I metals, including calcium, magnesium, and manganese, stabilize PAI-1 in the absence of vitronectin, whereas Type II metals, including cobalt, copper, and nickel, destabilize PAI-1 in the absence of vitronectin, but stabilize PAI-1 in its presence. To provide a mechanistic basis for understanding the unusual modulation of PAI-1 structure and activity, the binding characteristics and conformational effects of these two types of metals were further evaluated. Steady-state binding measurements using surface plasmon resonance indicated that both active and latent PAI-1 exhibit a dissociation constant in the low micromolar range for binding to immobilized nickel. Stopped-flow measurements of approach-to-equilibrium changes in intrinsic protein fluorescence indicated that the Type I and Type II metals bind in different modes that induce distinct conformational effects on PAI-1. Changes in the observed rate constants with varying concentrations of metal allowed accurate determination of binding affinities for cobalt, nickel, and copper, yielding dissociation constants of ∼40, 30, and 0.09 μM, respectively. Competition experiments that tested effects on PAI-1 stability were consistent with these measurements of affinity and indicate that copper binds tightly to PAI-1. PMID:21280128

  5. Impact of Mesenchymal Stem Cell secreted PAI-1 on colon cancer cell migration and proliferation

    SciTech Connect

    Hogan, Niamh M.; Joyce, Myles R.; Murphy, J. Mary; Barry, Frank P.; O’Brien, Timothy; Kerin, Michael J.; Dwyer, Roisin M.

    2013-06-14

    Highlights: •MSCs were directly co-cultured with colorectal cancer (CRC) cells on 3D scaffolds. •MSCs influence CRC protein/gene expression, proliferation and migration. •We report a significant functional role of MSC-secreted PAI-1 in colon cancer. -- Abstract: Mesenchymal Stem Cells are known to engraft and integrate into the architecture of colorectal tumours, with little known regarding their fate following engraftment. This study aimed to investigate mediators of Mesenchymal Stem Cell (MSC) and colon cancer cell (CCC) interactions. Mesenchymal Stem Cells and colon cancer cells (HT29 and HCT-116) were cultured individually or in co-culture on 3-dimensional scaffolds. Conditioned media containing all secreted factors was harvested at day 1, 3 and 7. Chemokine secretion and expression were analyzed by Chemi-array, ELISA (Macrophage migration inhibitory factor (MIF), plasminogen activator inhibitor type 1 (PAI-1)) and RQ-PCR. Colon cancer cell migration and proliferation in response to recombinant PAI-1, MSCs and MSCs + antibody to PAI-1 was analyzed using Transwell inserts and an MTS proliferation assay respectively. Chemi-array revealed secretion of a wide range of factors by each cell population, including PAI-1and MIF. ELISA analysis revealed Mesenchymal Stem Cells to secrete the highest levels of PAI-1 (MSC mean 10.6 ng/mL, CCC mean 1.01 ng/mL), while colon cancer cells were the principal source of MIF. MSC-secreted PAI-1 stimulated significant migration of both CCC lines, with an antibody to the chemokine shown to block this effect (67–88% blocking,). A cell-line dependant effect on CCC proliferation was shown for Mesenchymal Stem Cell-secreted PAI-1 with HCT-116 cells showing decreased proliferation at all concentrations, and HT29 cells showing increased proliferation in the presence of higher PAI-1 levels. This is the first study to identify PAI-1 as an important mediator of Mesenchymal Stem Cell/colon cancer cell interactions and highlights the

  6. The Role of TLR4 and Fyn Interaction on Lipopolysaccharide-Stimulated PAI-1 Expression in Astrocytes.

    PubMed

    Ko, Hyun Myung; Lee, Sung Hoon; Kim, Ki Chan; Joo, So Hyun; Choi, Wahn Soo; Shin, Chan Young

    2015-08-01

    Plasminogen activator inhibitor-1 (PAI-1) is an endogenous inhibitor of tissue plasminogen activator (tPA) that acts as a neuromodulator in various neurophysiological and pathological conditions. Several researchers including us reported the induction of PAI-1 during inflammatory condition; however, the mechanism regulating PAI-1 induction is not yet clear. In this study, we investigated the role of non-receptor tyrosine kinase Fyn in the regulation of lipopolysaccharide (LPS)-induced upregulation of PAI-1 in rat primary astrocyte. The activation of toll-like receptor 4 (TLR4) signaling, induced by its ligand LPS, stimulated a physical interaction between TLR4 and Fyn along with phosphorylation of tyrosine residue in both molecules as determined by co-immunoprecipitation experiments. Immunofluorescence staining also showed increased co-localization of TLR4-Fyn on cultured rat primary astrocytes after LPS treatment. The increased TRLR4-Fyn interaction induced expression of PAI-1 through the activation of PI3k/Akt/NFĸB pathway. Treatment with Src kinase inhibitor (PP2) or transfection of Fyn small interfering RNA (siRNA) into cultured rat primary astrocytes inhibited phosphorylation of tyrosine residue of TLR4 and blocked the interaction between TLR4 and Fyn resulting to the inhibition of LPS-induced expression of PAI-1. The activation of PI3K/Akt/NFĸB signaling cascades was also inhibited by Fyn knockdown in rat primary astrocytes. The induction of PAI-1 in rat primary astrocytes, which resulted in downregulation of tPA activity in culture supernatants, inhibited neurite outgrowth in cultured rat primary cortical neuron. The inhibition of neurite extension was prevented by PP2 or Fyn siRNA treatment in rat primary astrocytes. These results suggest the critical physiological role of TRL4-Fyn interaction in the modulation of PAI-1-tPA axis in astrocytes during neuroinflammatory responses such as ischemia/reperfusion injuries.

  7. Compression-induced HIF-1 enhances thrombosis and PAI-1 expression in mouse skin.

    PubMed

    Kaneko, Maki; Minematsu, Takeo; Yoshida, Mikako; Nishijima, Yoshimi; Noguchi, Hiroshi; Ohta, Yasunori; Nakagami, Gojiro; Mori, Taketoshi; Sanada, Hiromi

    2015-09-01

    Pressure ulcers result from tissue hypoxia caused by external forces. Thrombosis due to external forces is considered important, and hypoxia inducible factor-1 (HIF-1) is a master regulator of pressure ulcer development. To date, however, their causal relationship has not been determined. This study therefore investigated the mutual relationship between thrombosis and HIF-1 activation in compressed mouse skin, based on a hypothesis that HIF-1 regulation by plasminogen activator inhibitor-1 (PAI-1) enhances thrombosis. Compression of mouse skin significantly increased the numbers of thrombi and HIF-1α-positive cells compared with control skin. A thrombosis inhibitor significantly reduced the numbers of HIF-1α-positive cells and an HIF-1 inhibitor significantly inhibited thrombosis in compressed skin tissue, suggesting a mutual relationship between thrombosis and HIF-1 activation. Compression of mouse skin also enhanced the level of Pai-1 messenger RNA expression, but this increase was significantly reduced by treatment with an HIF-1 inhibitor, whereas a thrombosis inhibitor had no effect. These results suggested the involvement of PAI-1 in HIF-1-enhanced thrombosis and that an additional factor participates in regulating Pai-1 expression in compressed skin. These findings may suggest new strategies in pressure ulcer management.

  8. The gender-specific role of polymorphisms from the fibrinolytic, renin-angiotensin, and bradykinin systems in determining plasma t-PA and PAI-1 levels.

    PubMed

    Asselbergs, Folkert W; Williams, Scott M; Hebert, Patricia R; Coffey, Christopher S; Hillege, Hans L; Navis, Gerjan; Vaughan, Douglas E; van Gilst, Wiek H; Moore, Jason H

    2006-10-01

    Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) directly influence thrombus formation and degradation and thus risk for arterial thrombosis. We report here results from a genetic analysis of plasma t-PA and PAI-1 levels in a large population-based sample from the PREVEND study in Groningen, the Netherlands (n = 2,527). We measured polymorphisms from genes of the fibrinolytic system, the renin-angiotensin system (RAS), and the bradykinin system. We found that males had higher levels of natural-log transformed t-PA, and PAI-1 (P < 0.01) compared to females. When stratifying females by menopausal status, PAI-1 levels were only significantly different between pre-menopausal females and males (p < 0.001). Furthermore, we found that age, body mass index, and waist-to-hip ratio were significant predictors of t-PA and PAI-1 in both females and males, and that the regression relationships between these factors and plasma t-PA and PAI-1 were dependent on gender. In addition, we found that the PAI-1 4G/5G polymorphism was a significant predictor of PAI-1 levels in both females and males, that the angiotensin II type I receptor A1166C was a significant predictor of t-PA and PAI-1 levels in females, and that the bradykinin receptor B2 58CT polymorphism was a significant predictor of t-PA levels in females. In conclusion, this large population-based study showed that t-PA and PAI-1 levels are determined by several demographic and genetic factors involved in the fibrinolytic, RAS and bradykinin system. In addition, the results support the idea that the biology of t-PA and PAI-1 is different between females and males.

  9. Resistin regulates the expression of plasminogen activator inhibitor-1 in 3T3-L1 adipocytes.

    PubMed

    Ikeda, Yoshito; Tsuchiya, Hiroyuki; Hama, Susumu; Kajimoto, Kazuaki; Kogure, Kentaro

    2014-05-30

    Resistin and plasminogen activator inhibitor-1 (PAI-1) are adipokines, which are secreted from adipocytes. Increased plasma resistin and PAI-1 levels aggravate metabolic syndrome through exacerbation of insulin resistance and induction of chronic inflammation. However, the relationship between resistin and PAI-1 gene expression remains unclear. Previously, we found that resistin regulates lipid metabolism via carbohydrate responsive element-binding protein (ChREBP) during adipocyte maturation (Ikeda et al., 2013) [6]. In this study, to clarify the relationship between expression of resistin and PAI-1, PAI-1 expression in differentiated 3T3-L1 adipocytes was measured after transfection with anti-resistin siRNA. We found that PAI-1 gene expression and secreted PAI-1 protein were significantly decreased by resistin knockdown. Furthermore, phosphorylation of Akt, which can inhibit PAI-1 expression, was accelerated and the activity of protein phosphatase 2A (PP2A) was suppressed in resistin knockdown 3T3-L1 adipocytes. In addition, the expression of glucose transporter type 4, a ChREBP target gene, was reduced and was associated with inhibition of PP2A. The addition of culture medium collected from COS7 cells transfected with a resistin expression plasmid rescued the suppression of PAI-1 expression in resistin knockdown 3T3-L1 adipocytes. Our findings suggest that resistin regulates PAI-1 expression in 3T3-L1 adipocytes via Akt phosphorylation.

  10. The role of plasminogen activator inhibitor-1 in gastric mucosal protection

    PubMed Central

    Kenny, Susan; Steele, Islay; Lyons, Suzanne; Moore, Andrew R.; Murugesan, Senthil V.; Tiszlavicz, Laszlo; Dimaline, Rod; Pritchard, D. Mark; Varro, Andrea

    2013-01-01

    Gastric mucosal health is maintained in response to potentially damaging luminal factors. Aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) disrupt protective mechanisms leading to bleeding and ulceration. The plasminogen activator system has been implicated in fibrinolysis following gastric ulceration, and an inhibitor of this system, plasminogen activator inhibitor (PAI)-1, is expressed in gastric epithelial cells. In Helicobacter pylori-negative patients with normal gastric histology taking aspirin or NSAIDs, we found elevated gastric PAI-1 mRNA abundance compared with controls; the increase in patients on aspirin was independent of whether they were also taking proton pump inhibitors. In the same patients, aspirin tended to lower urokinase plasminogen activator mRNA. Immunohistochemistry indicated PAI-1 localization to epithelial cells. In a model system using MKN45 or AGS-GR cells transfected with a PAI-1 promoter-luciferase reporter construct, we found no evidence for upregulation of PAI-1 expression by indomethacin, and, in fact, cyclooxygenase products such as PGE2 and PGI2 weakly stimulated expression. Increased gastric PAI-1 mRNA was also found in mice following gavage with ethanol or indomethacin, but plasma PAI-1 was unaffected. In PAI-1−/− mice, gastric hemorrhagic lesions in response to ethanol or indomethacin were increased compared with C57BL/6 mice. In contrast, in PAI-1-H/Kβ mice in which PAI-1 is overexpressed in parietal cells, there were decreased lesions in response to ethanol and indomethacin. Thus, PAI-1 expression is increased in gastric epithelial cells in response to mucosal irritants such as aspirin and NSAIDs probably via an indirect mechanism, and PAI-1 acts as a local autoregulator to minimize mucosal damage. PMID:23494120

  11. Propofol treatment modulates neurite extension regulated by immunologically challenged rat primary astrocytes: a possible role of PAI-1.

    PubMed

    Ko, Hyun Myung; Joo, So Hyun; Lee, Sung Hoon; Kim, Hee Jin; Lee, Seung-Hyun; Cheong, Jae Hoon; Ryu, Jong Hoon; Kim, Jeong Min; Koo, Bon-Nyeo; Shin, Chan Young

    2015-04-01

    Propofol, a widely used anesthetic, regulates neurological processes including neurotoxicity, neuroprotection, glial activation, synaptic plasticity and neuronal maturation. Tissue plasminogen activator/tissue plasminogen activator inhibitor-1 (tPA/PAI-1) in CNS acts as a neuromodulator regulating synaptic plasticity, neurite outgrowth, seizure spreading and cell survival. Here, we investigated the effects of propofol on tPA/PAI-1 system using cultured neurons and astrocytes and their role in the regulation of neurite extension. Cultured rat primary astrocytes were treated with propofol (1-10 µM) and LPS (10 ng/ml). The expression of functional tPA/PAI-1 was examined by casein zymography, Western blot and RT-PCR. Alternatively, culture supernatants were added to cultured rat primary neuron to investigate the effects on neurite extension. Propofol alone did not affect tPA activity in rat primary cortical neuron. Similarly, propofol alone changed neither tPA nor PAI-1 activity in rat primary astrocytes. In immunologically challenged situation using LPS, propofol synergistically increased expression of PAI-1 in rat primary astrocytes without affecting tPA expression in a manner dependent on MAPKs activation. Increased expression of PAI-1 reduced tPA activity in LPS plus propofol-treated rat primary astrocytes. Consistent with the critical role of tPA activity in the regulation of neurite extension (Cho et al. 2013), the diminished tPA activity in astrocyte culture supernatants resulted in decreased neurite extension when administered to cultured rat primary cortical neuron. The results from the present study suggest that propofol, especially in immunologically-challenged situation, dysregulates tPA/PAI-1 system in brain. Whether the dysregulated tPA/PAI-1 activity adversely affects neural differentiation as well as regeneration of neuron in vivo should be empirically determined in the future.

  12. The HMG-CoA reductase inhibitor rosuvastatin inhibits plasminogen activator inhibitor-1 expression and secretion in human adipocytes.

    PubMed

    Laumen, Helmut; Skurk, Thomas; Hauner, Hans

    2008-02-01

    Human preadipocytes and adipocytes are known to produce the proatherogenic factor PAI-1 and proinflammatory cytokines, and obesity was found to be state of increased adipose production of these factors. In the present study, we investigated the effect of rosuvastatin on the regulation of PAI-1 gene expression in human adipocytes. Human preadipocytes, adipocytes in primary culture and the SGBS cell line were used as cell models. Cells were transfected using various constructs and promoter activity was measured as luciferase activity. PAI-1 expression was measured by quantitative RT-PCR and ELISA. Rosuvastatin inhibited PAI-1 mRNA expression and secretion of the protein in a concentration-dependent manner. This effect was reversed by isoprenoids. Addition of MEK-inhibitors and NFkappaB inhibitors also reduced PAI-1 expression and PAI-1 promoter luciferase activity. Further experiments revealed that rosuvastatin down-regulated the MEKK-1 mediated activation of the PAI-1 promoter. In conclusion our data suggest that rosuvastatin inhibits PAI-1 expression and release from human adipocytes via a MEKK-1-dependent but not a NFkappaB-dependent mechanism.

  13. HIF-2alpha-dependent PAI-1 induction contributes to angiogenesis in hepatocellular carcinoma.

    PubMed

    Geis, Theresa; Döring, Claudia; Popp, Rüdiger; Grossmann, Nina; Fleming, Ingrid; Hansmann, Martin-Leo; Dehne, Nathalie; Brüne, Bernhard

    2015-02-01

    Hypoxia promotes progression of hepatocellular carcinoma (HCC), not only affecting tumor cell proliferation and invasion, but also angiogenesis and thus, increasing the risk of metastasis. Hypoxia inducible factors (HIF)-1α and -2α cause adaptation of tumors to hypoxia, still with uncertainties towards the angiogenic switch. We created a stable knockdown of HIF-1α and HIF-2α in HepG2 cells and generated cocultures of HepG2 spheroids with embryonic bodies as an in vitro tumor model mimicking the cancer microenvironment. The naturally occuring oxygen and nutrient gradients within the cocultures allow us to question the role of distinct HIF isoforms in regulating HCC angiogenesis. In cocultures with a HIF-2α knockdown, angiogenesis was attenuated, while the knockdown of HIF-1α was without effect. Microarray analysis identified plasminogen activator inhibitor 1 (PAI-1) as a HIF-2α target gene in HepG2 cells. The knockdown of PAI-1 in HepG2 cells also lowered angiogenesis. Blocking plasmin, the downstream target of PAI-1, with aprotinin in HIF-2α knockdown (k/d) cells proved a cause-effect relation and restored angiogenesis, with no effect on control cocultures. Suggestively, HIF-2α increases PAI-1 to lower concentrations of active plasmin, thereby supporting angiogenesis. We conclude that the HIF-2α target gene PAI-1 favors the angiogenic switch in HCC. PMID:25489981

  14. HIF-2alpha-dependent PAI-1 induction contributes to angiogenesis in hepatocellular carcinoma.

    PubMed

    Geis, Theresa; Döring, Claudia; Popp, Rüdiger; Grossmann, Nina; Fleming, Ingrid; Hansmann, Martin-Leo; Dehne, Nathalie; Brüne, Bernhard

    2015-02-01

    Hypoxia promotes progression of hepatocellular carcinoma (HCC), not only affecting tumor cell proliferation and invasion, but also angiogenesis and thus, increasing the risk of metastasis. Hypoxia inducible factors (HIF)-1α and -2α cause adaptation of tumors to hypoxia, still with uncertainties towards the angiogenic switch. We created a stable knockdown of HIF-1α and HIF-2α in HepG2 cells and generated cocultures of HepG2 spheroids with embryonic bodies as an in vitro tumor model mimicking the cancer microenvironment. The naturally occuring oxygen and nutrient gradients within the cocultures allow us to question the role of distinct HIF isoforms in regulating HCC angiogenesis. In cocultures with a HIF-2α knockdown, angiogenesis was attenuated, while the knockdown of HIF-1α was without effect. Microarray analysis identified plasminogen activator inhibitor 1 (PAI-1) as a HIF-2α target gene in HepG2 cells. The knockdown of PAI-1 in HepG2 cells also lowered angiogenesis. Blocking plasmin, the downstream target of PAI-1, with aprotinin in HIF-2α knockdown (k/d) cells proved a cause-effect relation and restored angiogenesis, with no effect on control cocultures. Suggestively, HIF-2α increases PAI-1 to lower concentrations of active plasmin, thereby supporting angiogenesis. We conclude that the HIF-2α target gene PAI-1 favors the angiogenic switch in HCC.

  15. Effects of Pharmacological Inhibition and Genetic Deficiency of Plasminogen Activator Inhibitor-1 in Radiation-Induced Intestinal Injury

    SciTech Connect

    Abderrahmani, Rym; Francois, Agnes; Buard, Valerie; Benderitter, Marc; Sabourin, Jean-Christophe; Crandall, David L.; Milliat, Fabien

    2009-07-01

    Purpose: To investigate effects of plasminogen activator inhibitor 1 (PAI-1) genetic deficiency and pharmacological PAI-1 inhibition with PAI-039 in a mouse model of radiation-induced enteropathy. Methods and Materials: Wild-type (Wt) and PAI-1{sup -/-} knockout mice received a single dose of 19 Gy to an exteriorized localized intestinal segment. Sham and irradiated Wt mice were treated orally with 1 mg/g of PAI-039. Histological modifications were quantified using a radiation injury score. Moreover, intestinal gene expression was monitored by real-time PCR. Results: At 3 days after irradiation, PAI-039 abolished the radiation-induced increase in the plasma active form of PAI-1 and limited the radiation-induced gene expression of transforming growth factor {beta}1 (TGF-{beta}1), CTGF, PAI-1, and COL1A2. Moreover, PAI-039 conferred temporary protection against early lethality. PAI-039 treatment limited the radiation-induced increase of CTGF and PAI-1 at 2 weeks after irradiation but had no effect at 6 weeks. Radiation injuries were less severe in PAI-1{sup -/-} mice than in Wt mice, and despite the beneficial effect, 3 days after irradiation, PAI-039 had no effects on microscopic radiation injuries compared to untreated Wt mice. Conclusions: A genetic deficiency of PAI-1 is associated with amelioration of late radiation enteropathy. Pharmacological inhibition of PAI-1 by PAI-039 positively impacts the early, acute phase increase in plasma PAI-1 and the associated radiation-induced gene expression of inflammatory/extracellular matrix proteins. Since PAI-039 has been shown to inhibit the active form of PAI-1, as opposed to the complete loss of PAI-1 in the knockout animals, these data suggest that a PAI-1 inhibitor could be beneficial in treating radiation-induced tissue injury in acute settings where PAI-1 is elevated.

  16. Novel Combinatorial Therapeutic Targeting of PAI-1 (SERPINE1) Gene Expression in Alzheimer's Disease

    PubMed Central

    Kutz, Stacie M.; Higgins, Craig E.; Higgins, Paul J.

    2013-01-01

    Summary Accumulation of neurotoxic amyloid peptides (Aβ) in the brain, generated by β-site proteolytic processing of the amyloid precursor protein (APP), is the hallmark pathophysiologic feature of Alzheimer's disease. The plasmin-activating cascade, in which urokinase (uPA) and tissue-type (tPA) plasminogen activators convert plasminogen to the broad-spectrum protease plasmin, appears to serve a protective, Aβ-clearing, role in the central nervous system. Plasmin degrades Aβ and catalyzes α- site APP proteolysis generating nontoxic peptides. Plasmin activation in the brain is negatively regulated by the fast-acting clade E serine protease inhibitor (SERPIN) plasminogen activator inhibitor type-1 (PAI-1; SERPINE1) resulting in Aβ accumulation. PAI-1 and its major physiological inducer TGF-β1, moreover, are both increased in Alzheimer's disease models and implicated in the etiology and progression of human neurodegenerative disorders. Current findings support the hypothesis that targeting of PAI-1 function (by small molecule drugs) and/or gene expression (by histone deacetylase inhibitors) may constitute a clinically-relevant molecular approach to the therapy of neurodegenerative diseases associated with increased PAI-1 levels. PMID:23847772

  17. uPA and PAI-1-Related Signaling Pathways Differ between Primary Breast Cancers and Lymph Node Metastases12

    PubMed Central

    Malinowsky, Katharina; Wolff, Claudia; Berg, Daniela; Schuster, Tibor; Walch, Axel; Bronger, Holger; Mannsperger, Heiko; Schmidt, Christian; Korf, Ulrike; Höfler, Heinz; Becker, Karl-Friedrich

    2012-01-01

    The supporting role of urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor 1 (PAI-1) in migration and invasion is well known. In addition, both factors are key components in cancer cell-related signaling. However, little information is available for uPA and PAI-1-associated signaling pathways in primary cancers and corresponding lymph node metastases. The aim of this study was to compare the expression of uPA and PAI-1-associated signaling proteins in 52 primary breast cancers and corresponding metastases. Proteins were extracted from formalin-fixed paraffin-embedded tissue samples of the primary tumors and metastases. Protein lysates were subsequently analyzed by reverse phase protein array for the expression of members of the PI3K/AKT (FAK, GSK3-β, ILK, pGSK3-β, PI3K, and ROCK) and the MAPK pathways (pp38, pSTAT3, and p38). A solid correlation of uPA expression existed between primary tumors and metastases, whereas PAI-1 expression did not significantly correlate between them. The correlations of uPA and PAI-1 with signaling pathways found in primary tumors did not persist in metastases. Analysis of single molecules revealed that some correlated well between tumors and metastases (FAK, pGSK3-β, ILK, Met, PI3K, ROCK, uPA, p38, and pp38), whereas others did not (PAI-1 and GSK3-β). Whether the expression of a protein correlated between tumor and metastasis or not was independent of the pathway the protein is related to. These findings hint at a complete deregulation of uPA and PAI-1-related signaling in metastases, which might be the reason why uPA and PAI-1 reached clinical relevance only for lymph node-negative breast cancer tissues. PMID:22496926

  18. uPA and PAI-1-Related Signaling Pathways Differ between Primary Breast Cancers and Lymph Node Metastases.

    PubMed

    Malinowsky, Katharina; Wolff, Claudia; Berg, Daniela; Schuster, Tibor; Walch, Axel; Bronger, Holger; Mannsperger, Heiko; Schmidt, Christian; Korf, Ulrike; Höfler, Heinz; Becker, Karl-Friedrich

    2012-04-01

    The supporting role of urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor 1 (PAI-1) in migration and invasion is well known. In addition, both factors are key components in cancer cell-related signaling. However, little information is available for uPA and PAI-1-associated signaling pathways in primary cancers and corresponding lymph node metastases. The aim of this study was to compare the expression of uPA and PAI-1-associated signaling proteins in 52 primary breast cancers and corresponding metastases. Proteins were extracted from formalin-fixed paraffin-embedded tissue samples of the primary tumors and metastases. Protein lysates were subsequently analyzed by reverse phase protein array for the expression of members of the PI3K/AKT (FAK, GSK3-β, ILK, pGSK3-β, PI3K, and ROCK) and the MAPK pathways (pp38, pSTAT3, and p38). A solid correlation of uPA expression existed between primary tumors and metastases, whereas PAI-1 expression did not significantly correlate between them. The correlations of uPA and PAI-1 with signaling pathways found in primary tumors did not persist in metastases. Analysis of single molecules revealed that some correlated well between tumors and metastases (FAK, pGSK3-β, ILK, Met, PI3K, ROCK, uPA, p38, and pp38), whereas others did not (PAI-1 and GSK3-β). Whether the expression of a protein correlated between tumor and metastasis or not was independent of the pathway the protein is related to. These findings hint at a complete deregulation of uPA and PAI-1-related signaling in metastases, which might be the reason why uPA and PAI-1 reached clinical relevance only for lymph node-negative breast cancer tissues.

  19. Plasminogen Activator Inhibitor-1 in depression: Results from Animal and Clinical Studies.

    PubMed

    Jiang, Haitang; Li, Xiaoli; Chen, Suzhen; Lu, Na; Yue, Yingying; Liang, Jinfeng; Zhang, Zhijun; Yuan, Yonggui

    2016-01-01

    Evidence suggests that plasminogen activator inhibitor-1 (PAI-1) is a stress-related factor, and serum PAI-1 levels are increased in patients with major depressive disorders (MDD). Herein, we analysed PAI-1 protein levels in the brain, cerebrospinal fluid (CSF) and serum of rodents exposed to chronic unpredictable mild stress or treated with escitalopram. In addition, we examined PAI-1 concentrations in serum obtained from 17 drug-free depressed patients before and after escitalopram treatment. We found that PAI-1 expression was increased in area 1 of the cingulate cortex and prelimbic cortex of the medial prefrontal cortex as well as in the hippocampal cornu ammonis 3 and dentate gyrus in stressed rats. A downregulation of PAI-1 following chronic escitalopram treatment was also found. PAI-1 levels were higher in the CSF and serum in stressed rats than in controls, although the difference did not reach statistical significance in the serum. Escitalopram treatment significantly decreased PAI-1 levels in the serum, but not in the CSF. MDD patients had significantly greater serum PAI-1 concentrations than controls. Our results suggest that PAI-1 is implicated in the pathophysiology of depression. PMID:27456456

  20. Plasminogen Activator Inhibitor-1 in depression: Results from Animal and Clinical Studies

    PubMed Central

    Jiang, Haitang; Li, Xiaoli; Chen, Suzhen; Lu, Na; Yue, Yingying; Liang, Jinfeng; Zhang, Zhijun; Yuan, Yonggui

    2016-01-01

    Evidence suggests that plasminogen activator inhibitor-1 (PAI-1) is a stress-related factor, and serum PAI-1 levels are increased in patients with major depressive disorders (MDD). Herein, we analysed PAI-1 protein levels in the brain, cerebrospinal fluid (CSF) and serum of rodents exposed to chronic unpredictable mild stress or treated with escitalopram. In addition, we examined PAI-1 concentrations in serum obtained from 17 drug-free depressed patients before and after escitalopram treatment. We found that PAI-1 expression was increased in area 1 of the cingulate cortex and prelimbic cortex of the medial prefrontal cortex as well as in the hippocampal cornu ammonis 3 and dentate gyrus in stressed rats. A downregulation of PAI-1 following chronic escitalopram treatment was also found. PAI-1 levels were higher in the CSF and serum in stressed rats than in controls, although the difference did not reach statistical significance in the serum. Escitalopram treatment significantly decreased PAI-1 levels in the serum, but not in the CSF. MDD patients had significantly greater serum PAI-1 concentrations than controls. Our results suggest that PAI-1 is implicated in the pathophysiology of depression. PMID:27456456

  1. The c-myc-regulated gene mrl encodes plasminogen activator inhibitor 1.

    PubMed Central

    Prendergast, G C; Diamond, L E; Dahl, D; Cole, M D

    1990-01-01

    The DNA sequence of the c-myc-regulated gene mrl (G. C. Prendergast and M. D. Cole, Mol. Cell. Biol. 9:124-134, 1989) reveals that it encodes plasminogen activator inhibitor 1 (PAI-1), a regulator of extracellular proteolysis. Comparison of the human and mouse PAI-1 promoters and cDNA 3' noncoding regions revealed several highly conserved sequence domains, potential targets for c-myc and other factors influencing PAI-1 expression. We discuss possible roles for PAI-1 in normal and neoplastic cell growth control. PMID:2406566

  2. Inhibition of PAI-1 Via PAI-039 Improves Dermal Wound Closure in Diabetes.

    PubMed

    Rebalka, Irena A; Raleigh, Matthew J; D'Souza, Donna M; Coleman, Samantha K; Rebalka, Alexandra N; Hawke, Thomas J

    2015-07-01

    Diabetes impairs the ability to heal cutaneous wounds, leading to hospitalization, amputations, and death. Patients with diabetes experience elevated levels of plasminogen activator inhibitor 1 (PAI-1), regardless of their glycemic control. It has been demonstrated that PAI-1-deficient mice exhibit improved cutaneous wound healing, and that PAI-1 inhibition improves skeletal muscle repair in mice with type 1 diabetes mellitus, leading us to hypothesize that pharmacologically mediated reductions in PAI-1 using PAI-039 would normalize cutaneous wound healing in streptozotocin (STZ)-induced diabetic (STZ-diabetic) mice. To simulate the human condition of variations in wound care, wounds were aggravated or minimally handled postinjury. Following cutaneous injury, PAI-039 was orally administered twice daily for 10 days. Compared with nondiabetic mice, wounds in STZ-diabetic mice healed more slowly. Wound site aggravation exacerbated this deficit. PAI-1 inhibition had no effect on dermal collagen levels or wound bed size. PAI-039 treatment failed to improve angiogenesis in the wounds of STZ-diabetic mice and blunted angiogenesis in the wounds of nondiabetic mice. Importantly, PAI-039 treatment significantly improved epidermal cellular migration and wound re-epithelialization compared with vehicle-treated STZ-diabetic mice. These findings support the use of PAI-039 as a novel therapeutic agent to improve diabetic wound closure and demonstrate the primary mechanism of its action to be related to epidermal closure.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. Functionally stable plasminogen activator inhibitor-1 in a family with cardiovascular disease and vitiligo.

    PubMed

    Agirbasli, Mehmet; Eren, Mesut; Yasar, Songul; Delil, Kenan; Goktay, Fatih; Oner, Ebru Toksoy; Vaughan, Douglas E

    2014-07-01

    Vitiligo is a common skin condition with a complex pathophysiology characterized by the lack of pigmentation due to melanocyte degeneration. In this study, we investigated PAI-1 antigen (Ag) and activity levels in a 34 year old male with extensive vascular disease, alopecia areata and vitiligo. Fasting PAI-1 Ag and activity levels were measured at 9 a.m. in the subject and family members. Both PAI-1 Ag (67 ± 38 vs. 18.6 ± 6.5 ng/ml, P < 0.001) and specific activity (15.8 ± 10.0 vs. 7.6 ± 6.0 IU/pmol, P < 0.04) levels of PAI-1 were moderately elevated in subjects compared to the controls. PAI-1 kinetic studies demonstrated a markedly enhanced stability of plasma PAI-1 activity in the family members. Specific activity at 16 h was significantly higher than expected activity levels (0.078 ± 0.072 vs. 0.001 ± 0.001 IU/ng/ml, P < 0.001). While the exact mechanism of increased stability of PAI-1 activity in vitiligo is not known, it is likely due to post-translational modifications or increased binding affinity for a stabilizing cofactor. In conclusion, enhanced stability of PAI-1 may contribute to the pathophysiology of vascular disease and associated melanocyte degeneration. Systemic or local treatment with PAI-1 inhibitors may offer a potential treatment alternative to the near orphan status for vitiligo drug development. PMID:24197654

  5. Novel Plasminogen Activator Inhibitor-1 Inhibitors Prevent Diabetic Kidney Injury in a Mouse Model

    PubMed Central

    Park, Jong Hee; Lee, Jung Hwa; Lee, Hi Bahl; Miyata, Toshio; Ha, Hunjoo

    2016-01-01

    Diabetic nephropathy is the leading cause of end-stage renal disease worldwide, but no effective therapeutic strategy is available. Because plasminogen activator inhibitor-1 (PAI-1) is increasingly recognized as a key factor in extracellular matrix (ECM) accumulation in diabetic nephropathy, this study examined the renoprotective effects of TM5275 and TM5441, two novel orally active PAI-1 inhibitors that do not trigger bleeding episodes, in streptozotocin (STZ)-induced diabetic mice. TM5275 (50 mg/kg) and TM5441 (10 mg/kg) were administered orally for 16 weeks to STZ-induced diabetic and age-matched control mice. Relative to the control mice, the diabetic mice showed significantly increased (p < 0.05) plasma glucose and creatinine levels, urinary albumin excretion, kidney-to-bodyweight ratios, glomerular volume, and fractional mesangial area. Markers of fibrosis and inflammation along with PAI-1 were also upregulated in the kidney of diabetic mice, and treatment with TM5275 and TM5441 effectively inhibited albuminuria, mesangial expansion, ECM accumulation, and macrophage infiltration in diabetic kidneys. Furthermore, in mouse proximal tubular epithelial (mProx24) cells, both TM5275 and TM5441 effectively inhibited PAI-1-induced mRNA expression of fibrosis and inflammation markers and also reversed PAI-1-induced inhibition of plasmin activity, which confirmed the efficacy of the TM compounds as PAI-1 inhibitors. These data suggest that TM compounds could be used to prevent diabetic kidney injury. PMID:27258009

  6. Cell density-dependent nuclear accumulation of ELK3 is involved in suppression of PAI-1 expression.

    PubMed

    Tanaka, Shu; Nakao, Kazuyuki; Sekimoto, Toshihiro; Oka, Masahiro; Yoneda, Yoshihiro

    2013-07-01

    Cell-cell contact regulates the proliferation and differentiation of non-transformed cells, e.g., NIH/3T3 cells show growth arrest at high cell density. However, only a few reports described the dynamic behavior of transcription factors involved in this process. In this study, we showed that the mRNA levels of plasminogen activator inhibitor type 1 (PAI-1) decreased drastically at high cell density, and that ELK3, a member of the Ets transcription factor family, repressed PAI-1 expression. We also demonstrated that while ELK3 was distributed evenly throughout the cell at low cell density, it accumulated in the nucleus at high cell density, and that binding of DNA by ELK3 at the A domain facilitated its nuclear accumulation. Furthermore, we found that ETS1, a PAI-1 activator, occupied the ELK3-binding site within the PAI-1 promoter at low cell density, while it was released at high cell density. These results suggest that at high cell density, the switching of binding of transcription factors from ETS1 to ELK3 occurs at a specific binding site of the PAI-1 promoter, leading to the cell-density dependent suppression of PAI-1 expression.

  7. Antidepressant-like effect of n-3 PUFAs in CUMS rats: role of tPA/PAI-1 system.

    PubMed

    Tang, Mimi; Jiang, Pei; Li, Huande; Cai, Hualin; Liu, Yiping; Gong, Hui; Zhang, Lihong

    2015-02-01

    BDNF is strongly implicated in the development of depression. Recent evidence has indicated that tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) are related to the cleavage of pro-brain-derived neurotrophic factor (BDNF) into its mature form. Chronic unpredicted mild stress (CUMS) is widely used to induce depressive behaviors in rodents. Therefore, we investigated the effects of PUFAs and sertraline on tPA/PAI-1 system in CUMS rats. After 5 weeks of CUMS procedures, the rats were induced to a depressive-like state. The expressions of PAI-1 and proBDNF were increased in the prefrontal cortex and hippocampus of CUMS rats. N-3 polyunsaturated fatty acids (PUFAs) or sertraline administration reversed the changes in behavioral test and induced the expression of tPA in certain brain areas, but failed to restore the CUMS-induced PAI-1 expression. Meanwhile, the antidepressant treatment also accelerated the extracellular conversion of proBDNF into mature BDNF in CUMS rats. Our results firstly showed the synchronously altered balance of tPA/PAI-1 system in the prefrontal cortex and hippocampus of CUMS rats, which was partly ameliorated by PUFAs and sertraline medication, providing new evidence for the involvement of tPA/PAI-1 system in the progression and treatment of depression.

  8. PAI-1 synthesis in the human hepatoma cell line HepG2 is increased by cytokines--evidence that the liver contributes to acute phase behaviour of PAI-1.

    PubMed

    de Boer, J P; Abbink, J J; Brouwer, M C; Meijer, C; Roem, D; Voorn, G P; Lambers, J W; van Mourik, J A; Hack, C E

    1991-02-12

    The acute phase behaviour of the fast inhibitor of tissue-type plasminogen activator (PAI-1) in vivo has been attributed to increased synthesis by endothelial cells. However, most other acute phase proteins in vivo are synthesized in the liver, which process is regulated by cytokines and can be studied in the hepatoma derived cell line HepG2. In this study, we investigated whether the synthesis of PAI-1 by HepG2 cells is regulated by the cytokines recombinant IL-1, rIL-6 and rTNF. Recombinant IL-1 and rTNF each increased PAI-1 synthesis by HepG2 cells two to three fold, whereas rIL-6 hardly had an effect. Mixtures of rIL-1, rIL-6 and rTNF increased PAI-1 synthesis up to eleven fold. The effects observed were not due to non-specific effects on HepG2 cell metabolism, since synthesis of alpha-2-antiplasmin was not effected by any of those cytokines, whereas fibrinogen synthesis was increased three to four fold by rIL-6, but was unaffected by rIL-1. Thus, our results demonstrate that synthesis of PAI-1 by HepG2 cells is regulated by cytokines and implicate that the acute phase behaviour of PAI-1 in vivo at least in part may be due to an increased synthesis by the liver.

  9. Evaluating PAI-1 as a biomarker for stress in diving: human serum total PAI-1 is unaltered after 2 h dry exposures to 280 kPa hyperbaric air

    PubMed Central

    Eftedal, Ingrid; Fredriksen, Hallvard Aglen; Hjelde, Astrid; Møllerløkken, Andreas

    2015-01-01

    Plasminogen activator inhibitor (PAI-1) is induced in the vasculature and secreted into the vascular lumen in response to inflammation and oxidative stress. We have previously reported a fivefold increase in plasma PAI-1 from rats exposed to 708 kPa hyperbaric air. In the current study we assess the potential of human serum total PAI-1 as a biomarker for stress in compressed air diving. Eleven recreational divers, nine males and two females, completed four 2 h hyperbaric air exposures to 280 kPa in a pressure chamber over a period of 2 weeks. The air pressure corresponds to a diving depth of 18 m in water. Serum was collected before the study and again 3 h 30 min after completion of each hyperbaric exposure. All samples were taken in the afternoon to minimize the contribution of circadian variation. The analysis revealed no change in serum total PAI-1 after hyperbaric exposures within the group of divers (P = 0.064), but significant interindividual differences persisted throughout the study (P < 0.0005). A case of decompression sickness after the third round of hyperbaric exposure did not affect PAI-1. In conclusion, compressed air exposure to 280 kPa does not affect serum total PAI-1, and significant interindividual variation in PAI-1 levels may limit its usefulness as a biomarker. This does, however, not give a complete answer regarding PAI-1 in physiologically stressful dives. Further studies with different exposures and timing are needed for that. PMID:26109191

  10. Protumorigenic Activity of Plasminogen Activator Inhibitor-1 Through an Antiapoptotic Function

    PubMed Central

    2012-01-01

    Background Plasminogen activator inhibitor-1 (PAI-1) is a protease inhibitor but is paradoxically associated with poor outcomes in cancer patients. However, the mechanisms of its effects on tumor cells have not been explored. Methods Endogenous PAI-1 in human tumor cell lines (HT-1080, A549, HCT-116, and MDA-MB-231) was suppressed by small interfering RNAs (siRNAs) and PAI-039, a small molecule inhibitor of PAI-1, and the effects on apoptosis were examined. Tumorigenicity of PAI-1 knockdown (KD) tumor cells was examined in immunodeficient PAI-1 wild-type and knockout (KO) mice (9–15 per group), and event-free survival was analyzed by the Kaplan–Meier method. The effect of PAI-1 suppression on HT-1080 xenotransplanted tumors was evaluated for cell proliferation, apoptosis, and angiogenesis. All statistical tests were two-sided. Results Genetic and pharmacological inhibition of PAI-1 in the four tumor cell lines increased spontaneous apoptosis (mean fold increase relative to control: HT-1080, siRNA#1, mean = 4.0, 95% CI = 2.6 to 5.3, P < .001; siRNA#2, mean = 2.6, 95% CI = 2.4 to 2.9, P < .001, Student t test), which was blocked in the presence of recombinant PAI-1, a caspase-8 inhibitor, or Fas/FasL neutralizing antibodies and was partially attenuated by a plasmin inhibitor-aprotinin. PAI-1 KO mice implanted with PAI-1 KD HT-1080 cells had decreased tumorigenesis and prolonged survival compared with control mice (P = .002, log-rank test), and their tumors exhibited decreased cell proliferation and angiogenesis and increased apoptosis. Furthermore, five of 15 PAI-1 KO mice implanted with PAI-1 KD HT-1080 cells never developed tumors. Conclusions These data suggest that PAI-1 exerts a protective effect against tumor cell apoptosis by a mechanism that, in part, involves plasmin activation and inhibition of Fas/Fas-L-mediated apoptosis and may be a promising therapeutic target. PMID:22984202

  11. Interactive effect of PAI-1 4G/5G genotype and salt intake on PAI-1 antigen.

    PubMed

    Brown, N J; Murphey, L J; Srikuma, N; Koschachuhanan, N; Williams, G H; Vaughan, D E

    2001-06-01

    Activation of the renin-angiotensin-aldosterone system (RAAS) is associated with increased circulating PAI-1 antigen and increased risk of thrombotic cardiovascular events. A 4G/5G polymorphism located 675 bp upstream from the transcription start site of the PAI-1 gene affects PAI-1 antigen concentrations. To test the hypothesis that PAI-1 4G/5G genotype influences the effect of activation of the RAAS on PAI-1 expression, we measured morning PAI-1 antigen concentrations in 76 subjects with essential hypertension during low (10 mmol/d) and high (200 mmol/d) salt intake. Low salt intake was associated with activation of the RAAS as measured by plasma renin activity (2.3+/-0.2 versus 0.5+/-0.0 ng angiotensin I. mL(-1). h(-1), P<0.001) and aldosterone (529+/-40 versus 145+/-12 pmol/L). PAI-1 antigen concentrations were 17.9+/-2.7, 19.2+/-2.5, and 27.8+/-4.0 ng/mL during high salt intake and 19.2+/-2.7, 21.6+/-2.9, and 38.9+/-7.2 ng/mL during low salt intake in the 5G/5G (n=14), 4G/5G (n=40), and 4G/4G (n=22) groups, respectively. There was a significant effect of both salt intake (F=6.0, P=0.017) and PAI-1 4G/5G genotype (F=7.6, P=0.001) on PAI-1 antigen. More importantly, there was a significant interactive effect (F=7.8, P=0.001) of salt intake and PAI-1 4G/5G genotype on PAI-1 antigen. PAI-1 4G/5G genotype influenced the relationship between serum triglycerides and PAI-1 antigen such that the relationship was significant only in 4G homozygotes during either high (R(2)=0.31, P=0.014) or low (R(2)=0.37, P=0.006) salt intake. This study identifies an important gene-by-environment interaction that may influence cardiovascular morbidity and the response to pharmacological therapies that interrupt the RAAS.

  12. HIF-2alpha-dependent PAI-1 induction contributes to angiogenesis in hepatocellular carcinoma

    SciTech Connect

    Geis, Theresa; Döring, Claudia; Popp, Rüdiger; Grossmann, Nina; Fleming, Ingrid; Hansmann, Martin-Leo; Dehne, Nathalie; Brüne, Bernhard

    2015-02-01

    Hypoxia promotes progression of hepatocellular carcinoma (HCC), not only affecting tumor cell proliferation and invasion, but also angiogenesis and thus, increasing the risk of metastasis. Hypoxia inducible factors (HIF)-1α and -2α cause adaptation of tumors to hypoxia, still with uncertainties towards the angiogenic switch. We created a stable knockdown of HIF-1α and HIF-2α in HepG2 cells and generated cocultures of HepG2 spheroids with embryonic bodies as an in vitro tumor model mimicking the cancer microenvironment. The naturally occuring oxygen and nutrient gradients within the cocultures allow us to question the role of distinct HIF isoforms in regulating HCC angiogenesis. In cocultures with a HIF-2α knockdown, angiogenesis was attenuated, while the knockdown of HIF-1α was without effect. Microarray analysis identified plasminogen activator inhibitor 1 (PAI-1) as a HIF-2α target gene in HepG2 cells. The knockdown of PAI-1 in HepG2 cells also lowered angiogenesis. Blocking plasmin, the downstream target of PAI-1, with aprotinin in HIF-2α knockdown (k/d) cells proved a cause–effect relation and restored angiogenesis, with no effect on control cocultures. Suggestively, HIF-2α increases PAI-1 to lower concentrations of active plasmin, thereby supporting angiogenesis. We conclude that the HIF-2α target gene PAI-1 favors the angiogenic switch in HCC. - Highlights: • HepG2 were cocultured with stem cells to mimic a cancer microenvironment in vitro. • A knockdown of HIF-2α reduces angiogenesis. • PAI-1 was identified as a HIF-2α target gene in HCC by microarray analysis. • HIF-2α induces the angiogenic switch via inhibition of plasmin.

  13. Inhibitory effect of a new butadiene derivative on the production of plasminogen activator inhibitor-1 in cultured bovine endothelial cells.

    PubMed

    Ohtani, A; Takagi, T; Hirano, A; Murakami, J; Sasaki, Y

    1996-12-01

    Tissue-type plasminogen activator (t-PA) and its physiological inhibitor, plasminogen activator inhibitor-1 (PAI-1), are known to be synthesized by vascular endothelial cells and to play important roles in regulating the fibrinolytic activity of plasma. We found that a new butadiene derivative, (3E, 4E)-3-benzylidene-4-(3,4,5-trimethoxybenzylidene)pyrrolidine -2,5-dione (T-686), inhibits PAI-1 production without affecting plasminogen activator (PA) synthesis in cultured bovine endothelial cells. T-686 (1-10 microM) dose-dependently decreased the accumulation of PAI-1 in conditioned medium from the treated cells and elevated PA activity in the conditioned medium. Analysis of the conditioned medium by the zymography technique indicated that T-686 decreased the activities of PAI-1 with an M(r) of 55,000 and t-PA/PAI-1 complex with an M(r) of 99,000. Furthermore, T-686 attenuated the augmentation of PAI-1 antigen induced by lipopolysaccharide in the conditioned medium. The decrease of PAI-1 antigen was in parallel with the reduction of the PAI-1 mRNA level (Northern blots). These results suggest that T-686 can promote net fibrinolytic activity through suppression of PAI-1 production without affecting PA elaboration in endothelial cells.

  14. SIRT1-mediated epigenetic downregulation of plasminogen activator inhibitor-1 prevents vascular endothelial replicative senescence.

    PubMed

    Wan, Yan-Zhen; Gao, Peng; Zhou, Shuang; Zhang, Zhu-Qin; Hao, De-Long; Lian, Li-Shan; Li, Yong-Jun; Chen, Hou-Zao; Liu, De-Pei

    2014-10-01

    The inactivation of plasminogen activator inhibitor-1 (PAI-1) has been shown to exert beneficial effects in age-related vascular diseases. Limited information is available on the molecular mechanisms regarding the negatively regulated expression of PAI-1 in the vascular system. In this study, we observed an inverse correlation between SIRT1, a class III histone deacetylase, and PAI-1 expression in human atherosclerotic plaques and the aortas of old mice, suggesting that internal negative regulation exists between SIRT1 and PAI-1. SIRT1 overexpression reversed the increased PAI-1 expression in senescent human umbilical vein endothelial cells (HUVECs) and aortas of old mice, accompanied by decreased SA-β-gal activity in vitro and improved endothelial function and reduced arterial stiffness in vivo. Moreover, the SIRT1-mediated inhibition of PAI-1 expression exerted an antisenescence effect in HUVECs. Furthermore, we demonstrated that SIRT1 is able to bind to the PAI-1 promoter, resulting in a decrease in the acetylation of histone H4 lysine 16 (H4K16) on the PAI-1 promoter region. Thus, our findings suggest that the SIRT1-mediated epigenetic inhibition of PAI-1 expression exerts a protective effect in vascular endothelial senescence.

  15. SIRT1-mediated epigenetic downregulation of plasminogen activator inhibitor-1 prevents vascular endothelial replicative senescence

    PubMed Central

    Wan, Yan-Zhen; Gao, Peng; Zhou, Shuang; Zhang, Zhu-Qin; Hao, De-Long; Lian, Li-Shan; Li, Yong-Jun; Chen, Hou-Zao; Liu, De-Pei

    2014-01-01

    The inactivation of plasminogen activator inhibitor-1 (PAI-1) has been shown to exert beneficial effects in age-related vascular diseases. Limited information is available on the molecular mechanisms regarding the negatively regulated expression of PAI-1 in the vascular system. In this study, we observed an inverse correlation between SIRT1, a class III histone deacetylase, and PAI-1 expression in human atherosclerotic plaques and the aortas of old mice, suggesting that internal negative regulation exists between SIRT1 and PAI-1. SIRT1 overexpression reversed the increased PAI-1 expression in senescent human umbilical vein endothelial cells (HUVECs) and aortas of old mice, accompanied by decreased SA-β-gal activity in vitro and improved endothelial function and reduced arterial stiffness in vivo. Moreover, the SIRT1-mediated inhibition of PAI-1 expression exerted an antisenescence effect in HUVECs. Furthermore, we demonstrated that SIRT1 is able to bind to the PAI-1 promoter, resulting in a decrease in the acetylation of histone H4 lysine 16 (H4K16) on the PAI-1 promoter region. Thus, our findings suggest that the SIRT1-mediated epigenetic inhibition of PAI-1 expression exerts a protective effect in vascular endothelial senescence. PMID:25040736

  16. Genistein reduces tumor necrosis factor alpha-induced plasminogen activator inhibitor-1 transcription but not urokinase expression in human endothelial cells.

    PubMed

    van Hinsbergh, V W; Vermeer, M; Koolwijk, P; Grimbergen, J; Kooistra, T

    1994-11-01

    The plasminogen activator inhibitor PAI-1 is markedly elevated in vivo and in vitro upon exposure to the inflammatory mediators tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), and bacterial lipopolysaccharide. Here we report that the isoflavone compound genistein prevents the increase in synthesis of PAI-1 induced by these inflammatory mediators in human endothelial cells in vitro, and partially reduces the basal PAI-1 production by these cells. These effects of genistein were accompanied by a decrease in PAI-1 mRNA and in a suppression of the PAI-1 transcription rate as shown by run-on assay. A specific action of genistein, probably by inhibiting a tyrosine protein kinase, is likely, because the structural genistein analogue daidzein, which has a low tyrosine protein kinase inhibitor activity, did not inhibit PAI-1 synthesis. Vanadate, a tyrosine protein phosphatase inhibitor, increased PAI-1 production. The effect of genistein on PAI-1 synthesis was rather selective. Herbimycin A also reduced PAI-1 synthesis, but several other tyrosine protein kinase inhibitors, namely tyrphostin A47, methyl-2,5-dihydroxy-cinnamate, and compound 5, were unable to do so. All these tyrosine protein kinase inhibitors reduced basic fibroblast growth factor (b-FGF)-induced [3H]thymidine incorporation in endothelial cells. This indicates that the effect of genistein on PAI-1 transcription proceeds independently of its effect on mitogenesis. In contrast to TNF-alpha-induced PAI-1 production, the transcription and synthesis of urokinase-type plasminogen activator (u-PA) was not inhibited by genistein. A TNF-alpha-mutant (Trp32Thr86TNF alpha) that specifically recognizes the 55-kD TNF-receptor, mimicked the effects of TNF alpha on both PAI-1 and u-PA. Because genistein affected PAI-1, but not u-PA induced by this mutant, involvement of different TNF-receptors cannot underlie the difference in the effects of genistein on PAI-1 and u-PA synthesis. Because genistein also

  17. Cellular localization of type 1 plasminogen activator inhibitor messenger RNA and protein in murine renal tissue.

    PubMed Central

    Keeton, M.; Eguchi, Y.; Sawdey, M.; Ahn, C.; Loskutoff, D. J.

    1993-01-01

    Type 1 plasminogen activator inhibitor (PAI-1) may be markedly increased in the plasma of patients with endotoxemia and/or renal disease. To investigate renal PAI-1 production during acute endotoxemia, a murine model system was used. Mice were injected with either saline alone or saline containing 50 micrograms endotoxin, and sacrificed 3 hours later and their tissues analyzed for PAI-1 messenger RNA (mRNA) and antigen. Northern blot analysis confirmed that the level of renal PAI-1 mRNA was greatly increased in the endotoxemic mice relative to the saline controls. In situ hybridization was then performed to determine the cellular localization of PAI-1 mRNA within the renal tissues. In the control kidneys, low levels of PAI-1 mRNA were detected in the renal papilla and in the muscular walls of renal arteries. However, in the endotoxemic mice, an intense hybridization signal for PAI-1 mRNA was observed in glomerular and peritubular cells. These cells also stained positively for von Willebrand factor antigen, an endothelial cell-specific marker. The PAI-1 mRNA hybridization signal could further be observed in peritubular endothelial cells in the medulla and in endothelial cells of veins and arteries throughout the kidney. Immunochemical analysis revealed that PAI-1 antigen co-localized to the cytoplasm of cells expressing PAI-1 mRNA. This study provides the first direct evidence that PAI-1 is induced in endothelial cells of the kidney during endotoxemia in vivo and suggests a role for PAI-1 in the pathogenesis of renal disease. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8424466

  18. Association between plasminogen activator inhibitor-1 gene polymorphisms and recurrent pregnancy loss: a systematic review and meta-analysis.

    PubMed

    Chen, Hui; Nie, Shuping; Lu, Ming

    2015-04-01

    Human plasminogen activator inhibitor-1 (PAI-1) is closely related to embryonic development and pregnancy success. The association between PAI-1 gene polymorphisms (PAI-1-844G/A and PAI-1-675G/A) and the risk of recurrent pregnancy loss (RPL) is controversial. Therefore, we perform this review to clarify the association between PAI-1 gene polymorphisms and RPL risk. We performed a systematic search for studies that described the effect of PAI-1 polymorphisms on RPL risk. The odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were considered under recessive genetic models. Furthermore, we conducted a subgroup analysis based on the studies' geographic regions of origin. Data were analyzed using Stata 11.2 software. Eighteen studies were included, and a high degree of statistical heterogeneity existed among the studies. In this study, we found a significant association between the PAI-1-675G/A polymorphism and the risk of RPL under the recessive model (OR = 1.70, 95% CI = 1.21-2.38). However, no significant association between the PAI-1-844G/A polymorphism and RPL was noted. PAI-1-675G/A (4G/5G) polymorphisms play a potential role in RPL. The screening of PAI-1 (4G/5G) gene mutations should be included during an RPL diagnostic workup, and patients should be treated using anticoagulant therapy during pregnancy if necessary.

  19. Relationship between HMGB1 and PAI-1 after allogeneic hematopoietic stem cell transplantation

    PubMed Central

    Nomura, Shosaku; Maeda, Yoshinobu; Ishii, Kazuyoshi; Katayama, Yuta; Yagi, Hideo; Fujishima, Naoto; Ota, Shuichi; Moriyama, Masato; Ikezoe, Takayuki; Miyazaki, Yasuhiko; Hayashi, Kunio; Fujita, Shinya; Satake, Atsushi; Ito, Tomoki; Kyo, Taiichi; Tanimoto, Mitsune

    2016-01-01

    Background Conditioning regimens including total body irradiation (TBI) or cyclophosphamide can mobilize high-mobility group box 1 (HMGB1) to peripheral blood. Additionally, increased plasminogen activator inhibitor (PAI)-1 levels are associated with post-allogeneic hematopoietic stem cell transplantation (aHSCT). However, changes to circulating levels of HMGB1 after aHSCT are poorly understood. Materials and methods The study cohort included 289 patients who underwent aHSCT at one of 25 institutions in Japan. We have investigated the relationship between HMGB1 and PAI-1 following aHSCT. A significant increase in HMGB1 levels occurred after conditioning treatment. Additionally, levels of HMGB1 at day 0 were significantly increased in TBI+ patients and cyclophosphamide/TBI patients. Conclusion Our data revealed that an increased level of HMGB1 at day 0 following aHSCT correlates with increased PAI-1 after aHSCT, which is consistent with previous reports. Increased HMGB1 at day 0 after a conditioning regimen may play a role in transplantation-associated coagulopathy following aHSCT, because PAI-1 can accelerate procoagulant activity. PMID:26848281

  20. Binding of PAI-1 to endothelial cells stimulated by thymosin beta4 and modulation of their fibrinolytic potential.

    PubMed

    Boncela, Joanna; Smolarczyk, Katarzyna; Wyroba, Elzbieta; Cierniewski, Czeslaw S

    2006-01-13

    Our previous studies showed that thymosin beta4 (Tbeta4) induced the synthesis of plasminogen activator inhibitor-1 (PAI-1) in cultured human umbilical vein endothelial cells (HUVECs) via the AP-1 dependent mechanism and its enhanced secretion. In this work we provide evidence that the released PAI-1 is accumulated on the surface of HUVECs, exclusively in its active form, in a complex with alpha1-acid glycoprotein (AGP) that is also up-regulated and released from the cells. This mechanism is supported by several lines of experiments, in which expression of both proteins was analyzed by flow cytometry and their colocalization supported by confocal microscopy. PAI-1 did not bind to quiescent cells but only to the Tbeta4-activated endothelial cells. In contrast, significant amounts of AGP were found to be associated with the cells overexpressing enhanced green fluorescent protein (EGFP)-alpha1-acid glycoprotein (AGP) without Tbeta4 treatment. The AGP.PAI-1 complex was accumulated essentially at the basal surface of endothelial cells, and such cells showed (a) morphology characteristic for strongly adhered and spread cells and (b) significantly reduced plasmin formation. Taken together, these results provide the evidence supporting a novel mechanism by which active PAI-1 can be bound to the Tbeta4-activated endothelial cells, thus influencing their adhesive properties as well as their ability to generate plasmin.

  1. Plasminogen activator inhibitor-1 antisense oligodeoxynucleotides abrogate mesangial fibronectin accumulation.

    PubMed

    Park, Jehyun; Seo, Ji Yeon; Ha, Hunjoo

    2010-12-01

    Excessive extracellular matrix (ECM) accumulation is the main feature of chronic renal disease including diabetic nephropathy. Plasminogen activator inhibitor (PAI)-1 is known to play an important role in renal ECM accumulation in part through suppression of plasmin generation and matrix metalloproteinase (MMP) activation. The present study examined the effect of PAI-1 antisense oligodeoxynucleotide (ODN) on fibronectin upregulation and plasmin/MMP suppression in primary mesangial cells cultured under high glucose (HG) or transforming growth factor (TGF)-β1, major mediators of diabetic renal ECM accumulation. Growth arrested and synchronized rat primary mesangial cells were transfected with 1 µM phosphorothioate-modified antisense or control mis-match ODN for 24 hours with cationic liposome and then stimulated with 30 mM D-glucose or 2 ng/ml TGF-β1. PAI-1 or fibronectin protein was measured by Western blot analysis. Plasmin activity was determined using a synthetic fluorometric plasmin substrate and MMP-2 activity analyzed using zymography. HG and TGF-β1 significantly increased PAI-1 and fibronectin protein expression as well as decreased plasmin and MMP-2 activity. Transient transfection of mesangial cells with PAI-1 antisense ODN, but not mis-match ODN, effectively reversed basal as well as HG- and TGF-β1-induced suppression of plasmin and MMP-2 activity. Both basal and upregulated fibronectin secretion were also inhibited by PAI-1 antisense ODN. These data confirm that PAI-1 plays an important role in ECM accumulation in diabetic mesangium through suppression of protease activity and suggest that PAI-1 antisense ODN would be an effective therapeutic strategy for prevention of renal fibrosis including diabetic nephropathy.

  2. Effects of teneligliptin on PDMPs and PAI-1 in patients with diabetes on hemodialysis

    PubMed Central

    Okuda, Yoshinori; Omoto, Seitaro; Taniura, Takehito; Shouzu, Akira; Nomura, Shosaku

    2016-01-01

    Background Cardiovascular disease (CVD) is the main cause of death among hemodialysis (HD) patients. The effects of the dipeptidyl peptidase-4 inhibitor teneligliptin on CVD-related biomarkers in patients with type 2 diabetes mellitus (T2DM) receiving HD treatment are poorly understood. To determine whether teneligliptin has anti-CVD properties, we assessed its effects on soluble P-selectin (sP-selectin), platelet-derived microparticles (PDMPs), plasminogen activator inhibitor 1 (PAI-1), soluble E-selectin (sE-selectin), soluble vascular adhesion molecule 1 (sVCAM-1), and adiponectin plasma levels in HD and non-HD patients with T2DM. Methods Patients with T2DM eligible for teneligliptin monotherapy or combination therapy (eg, teneligliptin plus a sulfonylurea) were administered teneligliptin (20 mg/d) once daily for 6 months. Plasma levels of sP-selectin, PDMPs, PAI-1, sE-selectin, sVCAM-1, and adiponectin were measured by enzyme-linked immunosorbent assay at baseline and after 3 months and 6 months of treatment. Results Teneligliptin therapy significantly reduced plasma levels of sP-selectin, PDMPs, and PAI-1 compared with baseline levels, while significantly increasing adiponectin levels. sE-selectin and sVCAM-1 levels were significantly decreased only at 6 months. The reduction in sP-selectin, PDMPs, and PAI-1 was more significant in HD patients than in non-HD patients. However, the improvement in adiponectin levels was unchanged with HD treatment. Conclusion By modulating PDMPs or PAI-1, teneligliptin shows an antiatherothrombotic effect that may be beneficial in the primary prevention of CVD in patients with T2DM on HD. PMID:27110135

  3. Analysis of the inhibition of PAI-1 by metal theaflavin complexes and their degradation products.

    PubMed

    Jankun, Jerzy; Kondray, Victor; Skrzypczak-Jankun, Ewa

    2013-05-01

    The inhibition of elements of the plasminogen activator system [urokinase (uPA), tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1)] plays an important role in human diseases. PAI-1 is overexpressed in obesity and diabetes, and the inhibition of this protein has been postulated to alleviate the symptoms of both disorders. We found that two theaflavins (TFs) from black tea inhibit PAI-1 and we suggest that the beneficial effects of drinking tea may be associated with the suppression of PAI-1 activity by theaflavins. Epidemiological studies are controversial; some studies show the beneficial effects of drinking black tea on obesity and diabetes, while others do not. TFs, a family of compounds that can comprise up to 40% the dry weight of black tea, are responsible for the characteristic color, and they are known to chelate metals. We hypothesized that the content/variety of metals present in drinking water may be one of the reasons for such controversies in the population studies. TFs are excellent chelating compounds by trapping metals into complexes; thus, the quality of water used for tea preparation may influence changes in the formation of new products according to TF affinity for different metals, as well as their high molecular weight oxidation products. Our modeling and docking studies suggest that TF/metal complexes have similar affinity to PAI-1 as native TFs. However, analyses using liquid chromatography-mass spectroscopy (LC-MS) revealed the presence of TF degradation products in tea brewed using water containing metal salts. These can further form high molecular weight oxidation products. Thus, metals present in tea could diminish the beneficial effects of black tea by reducing TF concentration via metal-induced degradation and precipitation.

  4. Copper(II) Ions Increase Plasminogen Activator Inhibitor Type 1 Dynamics in Key Structural Regions That Govern Stability.

    PubMed

    Bucci, Joel C; Trelle, Morten Beck; McClintock, Carlee S; Qureshi, Tihami; Jørgensen, Thomas J D; Peterson, Cynthia B

    2016-08-01

    Plasminogen activator inhibitor type 1 (PAI-1) regulates the fibrinolysis pathway by inhibiting the protease activity of plasminogen activators. PAI-1 works in concert with vitronectin (VN), an extracellular protein that aids in localization of active PAI-1 to tissues. The Peterson laboratory demonstrated that Cu(II) and other transition metals modulate the stability of PAI-1, exhibiting effects that are dependent on the presence or absence of the somatomedin B (SMB) domain of VN. The study presented here dissects the changes in molecular dynamics underlying the destabilizing effects of Cu(II) on PAI-1. We utilize backbone amide hydrogen/deuterium exchange monitored by mass spectrometry to assess PAI-1 dynamics in the presence and absence of Cu(II) ions with and without the SMB domain of VN. We show that Cu(II) produces an increase in dynamics in regions important for the function and overall stability of PAI-1, while the SMB domain elicits virtually the opposite effect. A mutant form of PAI-1 lacking two N-terminal histidine residues at positions 2 and 3 exhibits similar increases in dynamics upon Cu(II) binding compared to that of active wild-type PAI-1, indicating that the observed structural effects are not a result of coordination of Cu(II) to these histidine residues. Finally, addition of Cu(II) results in an acceleration of the local unfolding kinetics of PAI-1 presumed to be on pathway to the latency conversion. The effect of ligands on the dynamics of PAI-1 adds another intriguing dimension to the mechanisms for regulation of PAI-1 stability and function. PMID:27416303

  5. Copper(II) Ions Increase Plasminogen Activator Inhibitor Type 1 Dynamics in Key Structural Regions That Govern Stability.

    PubMed

    Bucci, Joel C; Trelle, Morten Beck; McClintock, Carlee S; Qureshi, Tihami; Jørgensen, Thomas J D; Peterson, Cynthia B

    2016-08-01

    Plasminogen activator inhibitor type 1 (PAI-1) regulates the fibrinolysis pathway by inhibiting the protease activity of plasminogen activators. PAI-1 works in concert with vitronectin (VN), an extracellular protein that aids in localization of active PAI-1 to tissues. The Peterson laboratory demonstrated that Cu(II) and other transition metals modulate the stability of PAI-1, exhibiting effects that are dependent on the presence or absence of the somatomedin B (SMB) domain of VN. The study presented here dissects the changes in molecular dynamics underlying the destabilizing effects of Cu(II) on PAI-1. We utilize backbone amide hydrogen/deuterium exchange monitored by mass spectrometry to assess PAI-1 dynamics in the presence and absence of Cu(II) ions with and without the SMB domain of VN. We show that Cu(II) produces an increase in dynamics in regions important for the function and overall stability of PAI-1, while the SMB domain elicits virtually the opposite effect. A mutant form of PAI-1 lacking two N-terminal histidine residues at positions 2 and 3 exhibits similar increases in dynamics upon Cu(II) binding compared to that of active wild-type PAI-1, indicating that the observed structural effects are not a result of coordination of Cu(II) to these histidine residues. Finally, addition of Cu(II) results in an acceleration of the local unfolding kinetics of PAI-1 presumed to be on pathway to the latency conversion. The effect of ligands on the dynamics of PAI-1 adds another intriguing dimension to the mechanisms for regulation of PAI-1 stability and function.

  6. XR5967, a novel modulator of plasminogen activator inhibitor-1 activity, suppresses tumor cell invasion and angiogenesis in vitro.

    PubMed

    Brooks, Teresa D; Wang, Shouming W; Brünner, Nils; Charlton, Peter A

    2004-01-01

    Recent reports suggest that elevated levels of plasminogen activator inhibitor (PAI)-1 may contribute to tumor progression. We have recently shown that antibodies to PAI-1 block the invasive and migratory potential of human fibrosarcoma cells and suppress angiogenesis in vitro. Here we report the in vitro evaluation of a low-molecular-weight modulator of PAI-1, XR5967, on invasion, migration and angiogenesis. XR5967, a diketopiperazine, dose-dependently inhibited the activity of human and murine PAI-1, towards urokinase plasminogen activator (uPA), with IC50 values of 800 nM and 8.3 microM, respectively. This was confirmed by SDS-PAGE, revealing that XR5967 inhibited complex formation between PAI-1 and uPA. This suppression may be caused by XR5967 promoting insertion of the reactive center loop within PAI-1. XR5967 dose-dependently inhibited the invasion of human HT1080 fibrosarcoma cells through Matrigel. Their invasion was reduced by 57% (p<0.001) at 5 microM. HT1080 cell migration was inhibited in a similar manner, indicating that PAI-1 may play an additional role in invasion, which is distinct to its role in the regulation of proteolysis. The potential of XR5967 to inhibit the invasion/migration of human endothelial cells was investigated in an in vitro model of angiogenesis. In this model XR5967 reduced tubule formation by 77% at 5 microM (p<0.001), highlighting a crucial role for PAI-1 in angiogenesis. These data stress the importance of a balanced proteolysis in the processes of invasion, migration and angiogenesis. Our results support the clinical findings and indicate that modulation of PAI-1 activity, with low-molecular-weight inhibitor of PAI-1 activity, may be of therapeutic benefit for the treatment of cancer.

  7. High Incidence of ACE/PAI-1 in Association to a Spectrum of Other Polymorphic Cardiovascular Genes Involving PBMCs Proinflammatory Cytokines in Hypertensive Hypercholesterolemic Patients: Reversibility with a Combination of ACE Inhibitor and Statin

    PubMed Central

    Mouawad, Charbel; Haddad, Katia; Hamoui, Samar; Azar, Albert; Fajloun, Ziad; Makdissy, Nehman

    2015-01-01

    Cardiovascular diseases (CVDs) are significantly high in the Lebanese population with the two most predominant forms being atherosclerosis and venous thrombosis. The purpose of our study was to assess the association of a spectrum of CVD related genes and combined state of hypertension hypercholesterolemia (HH) in unrelated Lebanese. Twelve polymorphisms were studied by multiplex PCR and reverse hybridization of DNA from 171 healthy individuals and 144 HH subjects. Two genes were significantly associated with HH: ACE (OR: 9.20, P<0.0001) and PAI-1 (OR: 2.29, P = 0.007), respectively with the occurrence of the risky alleles “Del” and “4G”. The frequencies of the Del and 4G alleles were found to be 0.98 and 0.90 in the HH group versus 0.84 and 0.79 in the healthy group, respectively. Serum ACE activity and PAI-I increased significantly with Del/Del and 4G/5G genotypes. The co-expression of Del/4G(+/+) was detected in 113 out of 171 (66.0%) controls and 125 out of 144 (86.8%) HH subjects. Del/4G(-/-) was detected in only 6 (3.5%) controls and undetected in the HH group. Three venous thrombosis related genes [FV(Leiden), MTHFR(A1298C) and FXIII(V34L)] were significantly related to the prominence of the co-expression of Del/4G(+/+). A range of 2 to 8 combined polymorphisms co-expressed per subject where 5 mutations were the most detected. In Del/4G(+/+) subjects, peripheral blood mononuclear cells (PBMCs) produced significant elevated levels of IFN-γ and TNF-α contrary to IL-10, and no variations occurred for IL-4. ACE inhibitor (ramipril) in combination with statin (atorvastatin) and not alone reversed significantly the situation. This first report from Lebanon sheds light on an additional genetic predisposition of a complex spectrum of genes involved in CVD and suggests that the most requested gene FVL by physicians may not be sufficient to diagnose eventual future problems that can occur in the cardiovascular system. Subjects expressing the double mutations

  8. Role of plasminogen activator inhibitor-1 in senescence and aging.

    PubMed

    Eren, Mesut; Boe, Amanda E; Klyachko, Ekaterina A; Vaughan, Douglas E

    2014-09-01

    The average age of the US population continues to increase. Age is the most important determinant of disease and disability in humans, but the fundamental mechanisms of aging remain largely unknown. Many age-related diseases are associated with an impaired fibrinolytic system. Elevated plasminogen activator inhibitor-1 (PAI-1) levels are reported in age-associated clinical conditions including cardiovascular diseases, type 2 diabetes, obesity and inflammation. PAI-1 levels are also elevated in animal models of aging. While the association of PAI-1 with physiological aging is well documented, it is only recently that its critical role in the regulation of aging and senescence has become evident. PAI-1 is synthesized and secreted in senescent cells and contributes directly to the development of senescence by acting downstream of p53 and upstream of insulin-like growth factor binding protein-3. Pharmacologic inhibition or genetic deficiency of PAI-1 was shown to be protective against senescence and the aging-like phenotypes in kl/kl and N(ω)-nitro-l-arginine methyl ester-treated wild-type mice. Further investigation into PAI-1's role in senescence and aging will likely contribute to the prevention and treatment of aging-related pathologies.

  9. P38 MAPK inhibitors suppress biomarkers of hypertension end-organ damage, osteopontin and plasminogen activator inhibitor-1.

    PubMed

    Nerurkar, S S; Olzinski, A R; Frazier, K S; Mirabile, R C; O'Brien, S P; Jing, J; Rajagopalan, D; Yue, T L; Willette, R N

    2007-01-01

    The assessment of target organ damage is important in defining the optimal treatment of hypertension and blood pressure-related cardiovascular disease. The aims of the present study were (1) to investigate candidate biomarkers of target organ damage, osteopontin (OPN) and plasminogen activator inhibitor-1 (PAI-1), in models of malignant hypertension with well characterized end-organ pathology; and (2) to evaluate the effects of chronic treatment with a p38 MAPK inhibitor. Gene expression, plasma concentrations, and renal immunohistochemical localization of OPN and PAI-1 were measured in stroke-prone spontaneously hypertensive rats on a salt-fat diet (SFD SHR-SP) and in spontaneously hypertensive rats receiving N(omega)-nitro-L-arginine methyl ester (L-NAME SHR). Plasma concentrations of OPN and PAI-1 increased significantly in SFD SHR-SP and L-NAME SHR as compared with controls, (2.5-4.5-fold for OPN and 2.0-9.0-fold for PAI-1). The plasma levels of OPN and PAI-1 were significantly correlated with the urinary excretion of albumin (p < 0.0001). Elevations in urinary albumin, plasma OPN and PAI-1 were abolished by chronic treatment (4-8 weeks) with a specific p38 MAPK inhibitor, SB-239063AN. OPN immunoreactivity was localized predominantly in the apical portion of tubule epithelium, while PAI-1 immunoreactivity was robust in glomeruli, tubules and renal artery endothelium. Treatment with the p38 MAPK inhibitor significantly reduced OPN and PAI-1 protein expression in target organs. Kidney gene expression was increased for OPN (4.9- and 7.9-fold) and PAI-1 (2.8- and 11.5-fold) in SFD SHR-SP and L-NAME SHR, respectively. In-silico pathway analysis revealed that activation of p38 MAPK was linked to OPN and PAI-1 via SPI, c-fos and c-jun; suggesting that these pathways may play an important role in p38 MAPK-dependent hypertensive renal dysfunction. The results suggest that enhanced OPN and PAI-1 expression reflects end-organ damage in hypertension and that suppression

  10. Plasminogen activator inhibitor-1 deficiency ameliorates insulin resistance and hyperlipidemia but not bone loss in obese female mice.

    PubMed

    Tamura, Yukinori; Kawao, Naoyuki; Yano, Masato; Okada, Kiyotaka; Matsuo, Osamu; Kaji, Hiroshi

    2014-05-01

    We previously demonstrated that plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, is involved in type 1 diabetic bone loss in female mice. PAI-1 is well known as an adipogenic factor induced by obesity. We therefore examined the effects of PAI-1 deficiency on bone and glucose and lipid metabolism in high-fat and high-sucrose diet (HF/HSD)-induced obese female mice. Female wild-type (WT) and PAI-1-deficient mice were fed with HF/HSD or normal diet for 20 weeks from 10 weeks of age. HF/HSD increased the levels of plasma PAI-1 in WT mice. PAI-1 deficiency suppressed the levels of blood glucose, plasma insulin, and total cholesterol elevated by obesity. Moreover, PAI-1 deficiency improved glucose intolerance and insulin resistance induced by obesity. Bone mineral density (BMD) at trabecular bone as well as the levels of osterix, alkaline phosphatase, and receptor activator of nuclear factor κB ligand mRNA in tibia were decreased by HF/HSD in WT mice, and those changes by HF/HSD were not affected by PAI-1 deficiency. HF/HSD increased the levels of plasma TNF-α in both WT and PAI-1-deficient mice, and the levels of plasma TNF-α were negatively correlated with trabecular BMD in tibia of female mice. In conclusion, we revealed that PAI-1 deficiency does not affect the trabecular bone loss induced by obesity despite the amelioration of insulin resistance and hyperlipidemia in female mice. Our data suggest that the changes of BMD and bone metabolism by obesity might be independent of PAI-1 as well as glucose and lipid metabolism.

  11. Elevated levels of plasminogen activator inhibitor-1 in pulmonary edema fluid are associated with mortality in acute lung injury.

    PubMed

    Prabhakaran, Priya; Ware, Lorraine B; White, Kimberly E; Cross, Michael T; Matthay, Michael A; Olman, Mitchell A

    2003-07-01

    The alveolar fibrinolytic system is altered in acute lung injury (ALI). Levels of the fibrinolytic protease inhibitor, plasminogen activator inhibitor-1 (PAI-1), are too low in bronchoalveolar lavage to address its prognostic significance. This study was performed to assess whether PAI-1 antigen in undiluted pulmonary edema fluid levels can identify patients with ALI and predict their outcome. PAI-1 antigen levels in both plasma and edema fluid were higher in ALI compared with hydrostatic edema, and edema fluid PAI-1 values identified those with ALI with high sensitivity and specificity. Both the high plasma and edema fluid PAI-1 antigen values were associated with a higher mortality rate and fewer days of unassisted ventilation in patients with ALI. Differences in PAI-1 activity were concordant with levels of PAI-1 antigen. Although the fibrin-derived alveolar D-dimer levels were strikingly similar in both groups, ALI patients had a higher relative proportion of D-monomer. In conclusion, PAI-1 levels in edema fluid and plasma identify those with ALI that have a poor prognosis. The data indicate that fibrin turnover in early ALI is a consequence of a rapid fibrinogen influx and fractional fibrinolytic inhibition.

  12. Calcium regulation of tissue plasminogen activator and plasminogen activator inhibitor-1 release from cultured human vascular endothelial cells.

    PubMed

    Yamamoto, C; Kaji, T; Sakamoto, M; Kozuka, H; Koizumi, F

    1994-04-15

    Tissue plasminogen activator (t-PA) produced by vascular endothelial cells converts plasminogen to plasmin which degrades fibrin. Since t-PA activity is greatly potentiated in the presence of fibrin (1,2), the activator is implicated in intravascular fibrinolysis. On the other hand, endothelial cells also produce plasminogen activator inhibitor-1 (PAI-1) (3). The inhibitor associated with vascular endothelium rapidly inhibits t-PA, while that released into the liquid phase has a little anti-activator activity (4). However, clinical studies have shown that elevation of plasma PAI-1 level is a risk factor of thrombosis (5,6). It is thus suggested that the balance between t-PA and PAI-1 is important for the regulation of fibrinolysis. The release of t-PA and PAI-1 from vascular endothelial cells is regulated by physiological factors including thrombin (3,7), histamine (8), vasoconstrictor peptide endothelins (9,10) and cytokines (11). In addition, the regulation of the t-PA release and that of the PAI-1 release are not necessarily coupled. It has been shown that activated protein kinase C and cyclic AMP are involved in the stimulation and suppression, respectively, of the endothelial t-PA and PAI-1 production (12,13). However, the role of intracellular calcium in the regulation of endothelial t-PA and PAI-1 release has remained to be elucidated. In the present study, we investigated the effect of calcium ionophore A23187 on the release of t-PA antigen (t-PA:Ag) and PAI-1 antigen (PAI-1:Ag) from cultured vascular endothelial cells derived from human umbilical vein.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  14. Potential clinical relevance of uPA and PAI-1 levels in node-negative, postmenopausal breast cancer patients bearing histological grade II tumors with ER/PR expression, during an early follow-up.

    PubMed

    Buta, Marko; Džodić, Radan; Đurišić, Igor; Marković, Ivan; Vujasinović, Tijana; Markićević, Milan; Nikolić-Vukosavljević, Dragica

    2015-09-01

    We evaluated urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) prognostic value in postmenopausal, node-negative breast cancer patients bearing tumors with estrogen receptor (ER)/progesterone receptor (PR) expression, treated with locoregional therapy alone, within an early follow-up. We focused our analysis on tumors of histological grade II in order to improve its prognostic value and, consequently, to improve a decision-making process. The cytosol extracts of 73 tumor samples were used for assessing several biomarkers. ER and PR levels were measured by classical biochemical method. Cathepsin D was assayed by a radiometric immunoassay while both uPA and PAI-1 level determinations were performed by enzyme-linked immunosorbent assays. HER-2 gene amplification was determined by chromogenic in situ hybridization (CISH) in primary tumor tissue. Patients bearing tumors smaller than or equal to 2 cm (pT1) or those with low PAI-1 levels (PAI-1 < 6.35 pg/mg) showed favorable outcome compared to patients bearing tumors greater than 2 cm (pT2,3) or those with high PAI-1 levels, respectively. Analyses of 4 phenotypes, defined by tumor size and PAI-1 status, revealed that patients bearing either pT1 tumors, irrespective of PAI-1 levels, or pT2,3 tumors with low PAI-1 levels, had similar disease-free interval probabilities and showed favorable outcome compared to those bearing pT2,3 tumors with high PAI-1 levels. Our findings suggest that tumor size and PAI-1, used in combination as phenotypes are not only prognostic but might also be predictive in node-negative, postmenopausal breast cancer patients bearing histological grade II tumors with ER/PR expression, during an early follow-up period.

  15. The MAPK pathway and HIF-1 are involved in the induction of the human PAI-1 gene expression by insulin in the human hepatoma cell line HepG2.

    PubMed

    Dimova, Elitsa Y; Kietzmann, Thomas

    2006-12-01

    Enhanced levels of plasminogen activator inhibitor-1 (PAI-1) are considered to be a risk factor for pathological conditions associated with hypoxia or hyperinsulinemia. The expression of the PAI-1 gene is increased by insulin in different cells, although, the molecular mechanisms behind insulin-induced PAI-1 expression are not fully known yet. Here, we show that insulin upregulates human PAI-1 gene expression and promoter activity in HepG2 cells and that mutation of the hypoxia-responsive element (HRE)-binding hypoxia-inducible factor-1 (HIF-1) abolished the insulin effects. Mutation of E-boxes E4 and E5 abolished the insulin-dependent activation of the PAI-1 promoter only under normoxia, but did not affect it under hypoxia. Furthermore, the insulin effect was associated with activation of HIF-1alpha via mitogen-activated protein kinases (MAPKs) but not PDK1 and PKB in HepG2 cells. Furthermore, mutation of a putative FoxO1 binding site which was supposed to be involved in insulin-dependent PAI-1 gene expression influenced the insulin-dependent activation only under normoxia. Thus, insulin-dependent PAI-1 gene expression might be regulated by the action of both HIF-1 and FoxO1 transcription factors.

  16. Transforming growth factor-beta requires its target plasminogen activator inhibitor-1 for cytostatic activity.

    PubMed

    Kortlever, Roderik M; Nijwening, Jeroen H; Bernards, René

    2008-09-01

    The cytokine transforming growth factor beta (TGFbeta) has strong antiproliferative activity in most normal cells but contributes to tumor progression in the later stages of oncogenesis. It is not fully understood which TGFbeta target genes are causally involved in mediating its cytostatic activity. We report here that suppression of the TGFbeta target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference leads to escape from the cytostatic activity of TGFbeta both in human keratinocytes (HaCaTs) and primary mouse embryo fibroblasts. Consistent with this, PAI-1 knock-out mouse embryo fibroblasts are also resistant to TGFbeta growth arrest. Conversely, we show that ectopic expression of PAI-1 in proliferating HaCaT cells induces a growth arrest. PAI-1 knockdown does not interfere with canonical TGFbeta signaling as judged by SMAD phosphorylation and induction of bona fide TGFbeta target genes. Instead, knockdown of PAI-1 results in sustained activation of protein kinase B. Significantly, we find that constitutive protein kinase B activity leads to evasion of the growth-inhibitory action of TGFbeta. Our data are consistent with a model in which induction of PAI-1 by TGFbeta is critical for the induction of proliferation arrest.

  17. Alcoholic liver disease and the potential role of plasminogen activator inhibitor-1 and fibrin metabolism.

    PubMed

    Beier, Juliane I; Arteel, Gavin E

    2012-01-01

    Plasminogen activator inhibitor-1 (PAI-1) is a major player in fibrinolysis due to its classical role of inhibiting plasminogen activators. Although increased fibrinolysis is common in alcoholic cirrhosis, decreased fibrinolysis (driven mostly by elevated levels of PAI-1) is common during the development of alcoholic liver disease (ALD). However, whether or not PAI-1 plays a causal role in the development of early ALD was unclear. Recent studies in experimental models have suggested that PAI-1 may contribute to the development of early (steatosis), intermediate (steatohepatitis) and late (fibrosis) stages of ALD. For example, fatty liver owing to both acute and chronic ethanol was blunted by the genetic inhibition of PAI-1. This effect of targeting PAI-1 appears to be mediated, at least in part, by an increase in very low-density lipoprotein (VLDL) synthesis in the genetic absence of this acute phase protein. Results from a two-hit model employing ethanol and lipopolysaccharide administration suggest that PAI-1 plays a critical role in hepatic inflammation, most likely due to its ability to cause fibrin accumulation, which subsequently sensitizes the liver to ensuing damaging insults. Lastly, the role of PAI-1 in hepatic fibrosis is less clear and appears that PAI-1 may serve a dual role in this pathological change, both protective (enhancing regeneration) and damaging (blocking matrix degradation). In summary, results from these studies suggest that PAI-1 may play multiple roles in the various stages of ALD, both protective and damaging. The latter effect is mediated by its influence on steatosis (i.e. decreasing VLDL synthesis), inflammation (i.e. impairing fibrinolysis) and fibrosis (i.e. blunting matrix degradation), whereas the former is mediated by maintaining hepatocyte division after an injury.

  18. Alcoholic liver disease and the potential role of plasminogen activator inhibitor-1 and fibrin metabolism

    PubMed Central

    Beier, Juliane I; Arteel, Gavin E

    2016-01-01

    Plasminogen activator inhibitor-1 (PAI-1) is a major player in fibrinolysis due to its classical role of inhibiting plasminogen activators. Although increased fibrinolysis is common in alcoholic cirrhosis, decreased fibrinolysis (driven mostly by elevated levels of PAI-1) is common during the development of alcoholic liver disease (ALD). However, whether or not PAI-1 plays a causal role in the development of early ALD was unclear. Recent studies in experimental models have suggested that PAI-1 may contribute to the development of early (steatosis), intermediate (steatohepatitis) and late (fibrosis) stages of ALD. For example, fatty liver owing to both acute and chronic ethanol was blunted by the genetic inhibition of PAI-1. This effect of targeting PAI-1 appears to be mediated, at least in part, by an increase in very low-density lipoprotein (VLDL) synthesis in the genetic absence of this acute phase protein. Results from a two-hit model employing ethanol and lipopolysaccharide administration suggest that PAI-1 plays a critical role in hepatic inflammation, most likely due to its ability to cause fibrin accumulation, which subsequently sensitizes the liver to ensuing damaging insults. Lastly, the role of PAI-1 in hepatic fibrosis is less clear and appears that PAI-1 may serve a dual role in this pathological change, both protective (enhancing regeneration) and damaging (blocking matrix degradation). In summary, results from these studies suggest that PAI-1 may play multiple roles in the various stages of ALD, both protective and damaging. The latter effect is mediated by its influence on steatosis (i.e. decreasing VLDL synthesis), inflammation (i.e. impairing fibrinolysis) and fibrosis (i.e. blunting matrix degradation), whereas the former is mediated by maintaining hepatocyte division after an injury. PMID:22238286

  19. Gender differences in the relationships between plasma plasminogen activator inhibitor-1 activity and factors linked to the insulin resistance syndrome in essential hypertension.

    PubMed

    Toft, I; Bønaa, K H; Ingebretsen, O C; Nordøy, A; Birkeland, K I; Jenssen, T

    1997-03-01

    Impaired fibrinolysis due to elevated levels of plasma plasminogen activator inhibitor type 1 (PAI-1) is a risk factor for thromboembolic disease. Hypertension, obesity, derangements in lipid and glucose homeostasis, and elevated levels of PAI-1 are features of the insulin resistance syndrome. The interrelationships between PAI-1 and the metabolic disturbances seen in this condition are unsettled. We investigated the associations between PAI-1 activity and components of the insulin resistance syndrome in 53 men and 31 women with untreated hypertension. In men, PAI-1 activity correlated significantly with plasma glucose (r = .41, P = .002), insulin sensitivity (r = -.35, P = .01), and insulin-induced suppression of nonesterified fatty acid (NEFA) (r = -.43, P = .007). Plasma glucose and NEFA suppression were independently associated with PAI-1 activity in a multivariate analysis. In women, PAI-1 activity correlated with body mass index (r = .62, P = .0005), waist-to-hip ratio (r = .75, P = .0001), plasma glucose (r = .50, P = .007), insulin (r = .49, P = .009), proinsulin (r = .57, P = .002), C-peptide (r = .60, P = .0009), insulin sensitivity (r = -.74, P = .0001), NEFA suppression (r = -.64, P = .003), and triglycerides (r = .58, P = .001). In multivariate analyses, insulin sensitivity and NEFA suppression were independently associated with PAI-1 if waist-to-hip ratio was not included in the model. After introduction of waist-to-hip ratio into the model, waist-to-hip ratio was the only independent predictor of PAI-1 activity. We conclude that in women, waist-to-hip ratio, body mass index, and insulin-induced NEFA suppression are determinants for PAI-1 activity. In men, insulin-induced NEFA suppression and plasma glucose are independently associated with PAI-1 activity.

  20. Triglyceride concentration and waist circumference influence alcohol-related plasminogen activator inhibitor-1 activity increase in black South Africans.

    PubMed

    Pieters, Marlien; de Lange, Zelda; Hoekstra, Tiny; Ellis, Suria M; Kruger, Annamarie

    2010-12-01

    We investigated the association between alcohol consumption and plasminogen activator inhibitor-1 activity (PAI-1act) and fibrinogen concentration in a black South African population presenting with lower PAI-1act and higher fibrinogen than what is typically observed in white populations. We, furthermore, wanted to investigate the effect of urbanization, sex, central obesity, increased triglycerides, 4G/5G polymorphism (PAI-1 only) and BMI on the association of alcohol with PAI-1act and fibrinogen. Data from 2010 apparently healthy, randomly collected black South African volunteers from the Prospective Urban and Rural Epidemiological (PURE) study were cross-sectionally analyzed. Alcohol consumption was recorded using quantitative food frequency questionnaires and fasting blood samples were collected for biochemical analysis including PAI-1act and fibrinogen. Heavy alcohol consumption is associated with significantly increased PAI-1act, in the total population as well as in the women separately, and tended to be so in men. This alcohol-related PAI-1act increase was observed in volunteers with increased triglycerides and central obesity but not in volunteers with normal levels and waist circumference. Urbanization, the 4G/5G polymorphism and BMI did not affect the association of alcohol with PAI-1act. Moderate alcohol consumption is associated with decreased fibrinogen concentration. Sex and level of urbanization did not affect the association of alcohol with fibrinogen. Fibrinogen decreased in normal and overweight volunteers but not in obese and centrally obese volunteers following moderate alcohol consumption. Triglyceride levels and waist circumference influence alcohol-related PAI-1act increase potentially through modulating adipocyte and triglyceride-induced PAI-1 production. Obesity prevented alcohol-related fibrinogen decrease possibly by counteracting the anti-inflammatory effect of moderate alcohol consumption.

  1. Abnormal expression of plasminogen activator inhibitors in patients with gestational trophoblastic disease.

    PubMed Central

    Estellés, A.; Grancha, S.; Gilabert, J.; Thinnes, T.; Chirivella, M.; España, F.; Aznar, J.; Loskutoff, D. J.

    1996-01-01

    We previously reported significantly elevated levels of plasminogen activator inhibitor type 1 (PAI-1) in plasma and placenta from pregnant women with severe pre-eclampsia, and pre-eclampsia is a frequent problem in molar pregnancies. As increases in PAI-1 may contribute to the placental alterations that occur in pre-eclampsia, we have begun to investigate changes in PAI-1 as well as PAI-2 and several other components of the fibrinolytic system in patients with trophoblastic disease. Significant increases in plasma PAI-1 and decreases in plasma PAI-2 levels were observed in molar pregnancies when compared with the levels in normal pregnant women of similar gestational age. PAI-1 antigen levels also were increased, and PAI-2 levels were decreased in placenta from women with molar pregnancies compared with placenta obtained by spontaneous abortion. Immunohistochemical analysis revealed strong positive and specific staining of PAI-1 in trophoblastic epithelium in molar pregnancies and relatively weak staining of PAI-2. No association between the distribution of PAI-1 and vitronectin was found, and no specific signal for tissue type PA, urokinase type PA, tumor necrosis factor-alpha, or interleukin-1 was detected. In situ hybridization revealed an increase in PAI-1 but not PAI-2 mRNAs in placenta from molar pregnancies in comparison with placenta from abortions. These results demonstrate increased PAI-1 protein and mRNA in trophoblastic disease and suggest that localized elevated levels of PAI-1 may contribute to the hemostatic problems associated with this disorder. Images Figure 1 Figure 2 Figure 3 PMID:8863672

  2. The -844 G>A PAI-1 Polymorphism Is Associated with Acute Coronary Syndrome in Mexican Population

    PubMed Central

    García-González, Ilian Janet; Valle, Yeminia; Sandoval-Pinto, Elena; Valdés-Alvarado, Emmanuel; Valdez-Haro, Angélica; Francisco Muñoz-Valle, José; Flores-Salinas, Héctor Enrique; Figuera-Villanueva, Luis Eduardo; Dávalos-Rodríguez, Nory Omayra; Padilla-Gutiérrez, Jorge Ramón

    2015-01-01

    Background. Acute coronary syndrome (ACS) has an important impact in public health with high morbidity and mortality. Prothrombotic and proinflammatory states are involved in the pathogenesis of the disease. Plasminogen activator inhibitor-1 (PAI-1) is the major inhibitor of the fibrinolysis and also is part of immune response. The -844 G>A gene polymorphism is related to increased PAI-1 protein levels. The aim of the study is to evaluate the association of -844 G>A PAI-1 polymorphism with ACS. Methods. A total of 646 individuals were recruited from Western Mexico: 350 unrelated healthy subjects and 296 patients with diagnosis of ACS. Results. The most important risk factor in our population was hypertension, followed by smoking. The genetic distribution showed an association of the A allele (OR = 1.27, P = 0.04) and AA genotype (OR = 1.86, P = 0.02) with ACS. The recessive model displayed similar results (OR = 1.76, P = 0.02). As additional finding, we observed significant differences in the genetic distribution of ACS dyslipidemic patients (OR = 1.99, P = 0.04). The A allele and AA genotype of -844 polymorphism of PAI-1 gene are risk factors for ACS. The AA genotype might be associated with the development of dyslipidemia in ACS patients. PMID:25788758

  3. Accumulation of uPA-PAI-1 complexes inside the tumour cells is associated with axillary nodal invasion in progesterone-receptor-positive early breast cancer.

    PubMed

    Schneider, J; Pollán, M; Tejerina, A; Sánchez, J; Lucas, A R

    2003-01-13

    Both urokinase-like plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor (PAI-1), as well as uPA-PAI-1 complexes, have been identified as important prognostic factors in breast cancer. We have recently reported that the latter are identifiable inside breast cancer cells by means of immunohistochemistry. Using this technique, we have studied a series of 212 early (pT1) unifocal breast cancers and have correlated the expression of uPA-PAI-1 complexes, together with other clinical and biological features (histologic variety, histologic and nuclear grade, hormone receptors, Ki67 labelling index, c-erb-B2-, p53- and CD44std-expression) with or without the occurrence of axillary node invasion. In a logistic regression model, looking for associations with axillary metastasis, we found a statistically significant interaction between the presence of uPA-PAI-1 complexes and progesterone receptor positivity (P=0.04). A final model showed that the presence of uPA-PAI-1 complexes was a determinant factor for axillary metastasis among women carrying tumours expressing progesterone receptors. In these cases, the presence of uPA-PAI-1 complexes carried with it a nearly 14-fold risk of axillary node invasion (P=0.009). These results may indicate that small, hormone-receptor-positive breast cancers (with a theoretical good prognosis) may carry an elevated risk of nodal involvement if accumulation of uPA-PAI-1 complexes is shown inside their tumour cells by means of immunohistochemistry. PMID:12556966

  4. Epistatic interactions in genetic regulation of t-PA and PAI-1 levels in a Ghanaian population.

    PubMed

    Penrod, Nadia M; Poku, Kwabena A; Vaughan, Douglas E; Vaughn, Douglas E; Asselbergs, Folkert W; Brown, Nancy J; Moore, Jason H; Williams, Scott M

    2011-01-31

    The proteins, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1), act in concert to balance thrombus formation and degradation, thereby modulating the development of arterial thrombosis and excessive bleeding. PAI-1 is upregulated by the renin-angiotensin system (RAS), specifically by angiotensin II, the product of angiotensin converting enzyme (ACE) cleavage of angiotensin I, which is produced by the cleavage of angiotensinogen (AGT) by renin (REN). ACE indirectly stimulates the release of t-PA which, in turn, activates the corresponding fibrinolytic system. Single polymorphisms in these pathways have been shown to significantly impact plasma levels of t-PA and PAI-1 differently in Ghanaian males and females. Here we explore the involvement of epistatic interactions between the same polymorphisms in central genes of the RAS and fibrinolytic systems on plasma t-PA and PAI-1 levels within the same population (n = 992). Statistical modeling of pairwise interactions was done using two-way ANOVA between polymorphisms in the ETNK2, RENIN, ACE, PAI-1, t-PA, and AGT genes. The most significant interactions that associated with t-PA levels were between the ETNK2 A6135G and the REN T9435C polymorphisms in females (p = 0.006) and the REN T9435C and the TPA I/D polymorphisms (p = 0.005) in males. The most significant interactions for PAI-1 levels were with REN T9435C and the TPA I/D polymorphisms (p = 0.001) in females, and the association of REN G6567T with the TPA I/D polymorphisms (p = 0.032) in males. Our results provide evidence for multiple genetic effects that may not be detected using single SNP analysis. Because t-PA and PAI-1 have been implicated in cardiovascular disease these results support the idea that the genetic architecture of cardiovascular disease is complex. Therefore, it is necessary to consider the relationship between interacting polymorphisms of pathway specific genes that predict t-PA and PAI-1 levels.

  5. Relationship of hepatic and peripheral insulin resistance with plasminogen activator inhibitor-1 in Pima Indians.

    PubMed

    Nagi, D K; Tracy, R; Pratley, R

    1996-10-01

    Plasminogen activator inhibitor-1 (PAI-1) is related to insulin resistance and several components of the insulin resistance syndrome, and PAI-1 levels are elevated in subjects with non-insulin-dependent diabetes mellitus. Many Pima Indians are obese, insulin-resistant, and hyperinsulinemic, and they have high rates of diabetes but a low risk of ischemic heart disease. In contrast to whites and Asians, PAI-1 activity is similar between nondiabetic and diabetic Pima Indians. We therefore examined the association of PAI-1 with hepatic and peripheral insulin action measured using the hyperinsulinemic-euglycemic clamp. To investigate if insulin per se has any effect on PAI-1 in vivo, we also assessed the effects of endogenous (during a 75-g oral glucose load) and exogenous (during hyperinsulinemic clamp) insulin on PAI-1 antigen. Twenty-one (14 men and seven women; mean age, 26.3 +/- 4.8 years) Pima Indians underwent a 75-g oral glucose tolerance test (OGTT) and a sequential hyperinsulinemic-euglycemic clamp. Peripheral insulin action was measured as absolute glucose uptake (M value) and normalized to estimated metabolic body size (EMBS). Hepatic insulin action was measured as percent suppression of basal hepatic glucose output during hyperinsulinemia. PAI-1 antigen was determined using a two-site enzyme-linked immunosorbent assay that detects only free PAI-1. PAI-1 antigen concentrations were significantly related to body mass index ([BMI] rs = .54, P = .012), waist (rs=.52, P=.016) and thigh (rs=.63, P=.002) circumference, and fasting plasma insulin concentration (rs=.59, P=.004). PAI-1 antigen concentrations were not significantly associated with peripheral glucose uptake (M value) during either low-dose (rs= -.01, P=NS) or high-dose (rs= -.11, P=NS) insulin infusion. PAI-1 antigen was negatively correlated with basal hepatic glucose output (rs= -.57, P=.013) and percent suppression of hepatic glucose output during hyperinsulinemia (rs= -.69, P=.005). However, this

  6. Statins Increase Plasminogen Activator Inhibitor Type 1 Gene Transcription through a Pregnane X Receptor Regulated Element

    PubMed Central

    Stanley, Frederick M.; Linder, Kathryn M.; Cardozo, Timothy J.

    2015-01-01

    Plasminogen activator inhibitor type 1 (PAI-1) is a multifunctional protein that has important roles in inflammation and wound healing. Its aberrant regulation may contribute to many disease processes such as heart disease. The PAI-1 promoter is responsive to multiple inputs including cytokines, growth factors, steroids and oxidative stress. The statin drugs, atorvastatin, mevastatin and rosuvastatin, increased basal and stimulated expression of the PAI-1 promoter 3-fold. A statin-responsive, nuclear hormone response element was previously identified in the PAI-1 promoter, but it was incompletely characterized. We characterized this direct repeat (DR) of AGGTCA with a 3-nucleotide spacer at -269/-255 using deletion and directed mutagenesis. Deletion or mutation of this element increased basal transcription from the promoter suggesting that it repressed PAI-1 transcription in the unliganded state. The half-site spacing and the ligand specificity suggested that this might be a pregnane X receptor (PXR) responsive element. Computational molecular docking showed that atorvastatin, mevastatin and rosuvastatin were structurally compatible with the PXR ligand-binding pocket in its agonist conformation. Experiments with Gal4 DNA binding domain fusion proteins showed that Gal4-PXR was activated by statins while other DR + 3 binding nuclear receptor fusions were not. Overexpression of PXR further enhanced PAI-1 transcription in response to statins. Finally, ChIP experiments using Halo-tagged PXR and RXR demonstrated that both components of the PXR-RXR heterodimer bound to this region of the PAI-1 promoter. PMID:26379245

  7. Crystal structure of plasminogen activator inhibitor-1 in an active conformation with normal thermodynamic stability.

    PubMed

    Jensen, Jan K; Thompson, Lawrence C; Bucci, Joel C; Nissen, Poul; Gettins, Peter G W; Peterson, Cynthia B; Andreasen, Peter A; Morth, J Preben

    2011-08-26

    The serpin plasminogen activator inhibitor-1 (PAI-1) is a crucial regulator in fibrinolysis and tissue remodeling. PAI-1 has been associated with several pathological conditions and is a validated prognostic marker in human cancers. However, structural information about the native inhibitory form of PAI-1 has been elusive because of its inherent conformational instability and rapid conversion to a latent, inactive structure. Here we report the crystal structure of PAI-1 W175F at 2.3 Å resolution as the first model of the metastable native molecule. Structural comparison with a quadruple mutant (14-1B) previously used as representative of the active state uncovered key differences. The most striking differences occur near the region that houses three of the four mutations in the 14-1B PAI-1 structure. Prominent changes are localized within a loop connecting β-strand 3A with the F helix, in which a previously observed 3(10)-helix is absent in the new structure. Notably these structural changes are found near the binding site for the cofactor vitronectin. Because vitronectin is the only known physiological regulator of PAI-1 that slows down the latency conversion, the structure of this region is important. Furthermore, the previously identified chloride-binding site close to the F-helix is absent from the present structure and likely to be artifactual, because of its dependence on the 14-1B mutations. Instead we found a different chlorine-binding site that is likely to be present in wild type PAI-1 and that more satisfactorily accounts for the chlorine stabilizing effect on PAI-1.

  8. Crystal Structure of Plasminogen Activator Inhibitor-1 in an Active Conformation with Normal Thermodynamic Stability*

    PubMed Central

    Jensen, Jan K.; Thompson, Lawrence C.; Bucci, Joel C.; Nissen, Poul; Gettins, Peter G. W.; Peterson, Cynthia B.; Andreasen, Peter A.; Morth, J. Preben

    2011-01-01

    The serpin plasminogen activator inhibitor-1 (PAI-1) is a crucial regulator in fibrinolysis and tissue remodeling. PAI-1 has been associated with several pathological conditions and is a validated prognostic marker in human cancers. However, structural information about the native inhibitory form of PAI-1 has been elusive because of its inherent conformational instability and rapid conversion to a latent, inactive structure. Here we report the crystal structure of PAI-1 W175F at 2.3 Å resolution as the first model of the metastable native molecule. Structural comparison with a quadruple mutant (14-1B) previously used as representative of the active state uncovered key differences. The most striking differences occur near the region that houses three of the four mutations in the 14-1B PAI-1 structure. Prominent changes are localized within a loop connecting β-strand 3A with the F helix, in which a previously observed 310-helix is absent in the new structure. Notably these structural changes are found near the binding site for the cofactor vitronectin. Because vitronectin is the only known physiological regulator of PAI-1 that slows down the latency conversion, the structure of this region is important. Furthermore, the previously identified chloride-binding site close to the F-helix is absent from the present structure and likely to be artifactual, because of its dependence on the 14-1B mutations. Instead we found a different chlorine-binding site that is likely to be present in wild type PAI-1 and that more satisfactorily accounts for the chlorine stabilizing effect on PAI-1. PMID:21697084

  9. Are free fatty acids related to plasma plasminogen activator inhibitor 1 in android obesity?

    PubMed

    Bastard, J P; Bruckert, E; Robert, J J; Ankri, A; Grimaldi, A; Jardel, C; Hainque, B

    1995-11-01

    Plasminogen activator inhibitor 1 (PAI-1) levels are elevated in obese insulin-resistant subjects. However the mechanism underlying increased PAI-1 levels is unknown. To determine the impact of diabetes on PAI-1 levels and its possible relationship to insulin resistance, hyperinsulinemic euglycemic clamp studies were performed in nine lean control subjects, nine non-diabetic obese subjects and eight obese patients with NIDDM. Fasting plasma PAI-1 levels were 4.0 to 4.7 fold higher in the two obese groups than in the control group. During the 40 mU/m2 x min insulin infusion, suppression of FFA concentration was correlated with fasting plasma PAI-1 levels in both obese non-diabetic and obese NIDDM subjects. It is concluded that (1) obesity rather than diabetes itself plays a major role for the increased PAI-1 levels in NIDDM; (2) resistance to the antilipolytic effect of insulin, resulting in increased FFA concentrations, may participate in producing elevated PAI-1 levels in android obese subjects.

  10. Beta-Adrenoceptor agonists and other cAMP elevating agents suppress PAI-1 production of human adipocytes in primary culture.

    PubMed

    Gottschling-Zeller, H; Aprath, I; Skurk, T; Hauner, H

    2000-01-01

    Recent studies showed that catecholamines contribute to the regulation of plasminogen activator inhibitor-1 (PAI-1) expression, at least in endothelial cells. Aim of this study was to examine the role of catecholamines on PAI-1 production by human adipocytes and, in particular, to clarify which adrenoceptor (AR) subtypes are involved. Addition of the unselective AR agonist isoproterenol led to a dose- and time-dependent suppression of PAI-1 mRNA and protein release in adipocytes from the subcutaneous and omental depot of obese subjects. A similar degree of suppression was observed in subcutaneous mammary adipocytes of lean women. This effect was mainly mediated via the beta2-adrenoceptor according to experiments using selective agonists. Moreover, addition of cAMP-elevating agents such as dibutyryl-cAMP, forskolin and the phosphodiesterase inhibitors isobutyl-methylxanthine and milrinone resulted in a reduction of PAI-1 of varying degrees. In conclusion, the results of this study support the assumption that catecholamines are able to down-regulate PAI-1 expression and secretion in human adipocytes via beta-adrenergic receptors.

  11. Upstream stimulatory factor-2 mediates quercetin-induced suppression of PAI-1 gene expression in human endothelial cells.

    PubMed

    Olave, Nélida C; Grenett, Maximiliano H; Cadeiras, Martin; Grenett, Hernan E; Higgins, Paul J

    2010-10-15

    The polyphenol quercetin (Quer) represses expression of the cardiovascular disease risk factor plasminogen activator inhibitor-1 (PAI-1) in cultured endothelial cells (ECs). Transfection of PAI-1 promoter-luciferase reporter deletion constructs identified a 251-bp fragment (nucleotides -800 to -549) responsive to Quer. Two E-box motifs (CACGTG), at map positions -691 (E-box1) and -575 (E-box2), are platforms for occupancy by several members of the c-MYC family of basic helix-loop-helix leucine zipper (bHLH-LZ) proteins. Promoter truncation and electrophoretic mobility shift/supershift analyses identified upstream stimulatory factor (USF)-1 and USF-2 as E-box1/E-box2 binding factors. ECs co-transfected with a 251 bp PAI-1 promoter fragment containing the two E-box motifs (p251/luc) and a USF-2 expression vector (pUSF-2/pcDNA) exhibited reduced luciferase activity versus p251/luc alone. Overexpression of USF-2 decreased, while transfection of a dominant-negative USF construct increased, EC growth consistent with the known anti-proliferative properties of USF proteins. Quer-induced decreases in PAI-1 expression and reduced cell proliferation may contribute, at least in part, to the cardioprotective benefit associated with daily intake of polyphenols.

  12. Mechanism of Plasminogen Activator Inhibitor-1 regulation by Oncostatin M and Interleukin-1 in human astrocytes

    PubMed Central

    Kasza, Aneta; Kiss, Daniel L.; Gopalan, Sunita; Xu, Weili; Rydel, Russell E.; Koj, Aleksander; Kordula, Tomasz

    2015-01-01

    Glial cells that produce and respond to various cytokines mediate inflammatory processes in the brain. Here, we show that oncostatin M (OSM) and interleukin-1 (IL-1) regulate the expression of plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) in human astrocytes. Using the PAI-1 reporter constructs we show that the −58 to −51 proximal element mediates activation by both cytokines. This element is already bound by c-fos/c-jun heterodimers in unstimulated astrocytes, and treatment with cytokine strongly stimulates both expression of c-fos and binding of c-fos/c-jun heterodimers. In addition, IL-1 activates an inhibitory mechanism that downregulates PAI-1 expression after longer exposure to this cytokine. Overexpression of dominant-negative signal transducer and activator of transcription-1 (STAT1), STAT3, STAT5 and inhibitor of nuclear factor kB (IkB) suppressed OSM/IL-1-induced expression of the PAI-1 reporter construct. We conclude that OSM and IL-1 regulate the PAI-1 gene expression via up-regulating c-fos levels and subsequent binding of c-fos/c-jun heterodimers to the proximal element of the PAI-1 gene. PMID:12390531

  13. Plasminogen activator inhibitor-1 4G/5G polymorphism, factor V Leiden, prothrombin mutations and the risk of VTE recurrence.

    PubMed

    Sundquist, Kristina; Wang, Xiao; Svensson, Peter J; Sundquist, Jan; Hedelius, Anna; Larsson Lönn, Sara; Zöller, Bengt; Memon, Ashfaque A

    2015-11-25

    Plasminogen-activator inhibitor (PAI)-1 is an important inhibitor of the plasminogen/plasmin system. PAI-1 levels are influenced by the 4G/5G polymorphism in the PAI-1 promoter. We investigated the relationship between the PAI-1 polymorphism and VTE recurrence, and its possible modification by factor V Leiden (FVL) and prothrombin (PTM) mutations. Patients (n=1,069) from the Malmö Thrombophilia Study were followed from discontinuation of anticoagulant treatment until diagnosis of VTE recurrence or the end of the study (maximum follow-up 9.8 years). One hundred twenty-seven patients (11.9 %) had VTE recurrence. PAI-1 was genotyped by TaqMan PCR. Cox regression analysis adjusted for age, sex and acquired risk factors of VTE showed no evidence of an association between PAI-1 genotype and risk of VTE recurrence in the study population as a whole. However, by including an interaction term in the analysis we showed that FVL but not PTM modified the effect of PAI-1 genotype: patients with the 4G allele plus FVL had a higher risk of VTE recurrence [hazard ratio (HR) =2.3, 95 % confidence interval (CI) =1.5-3.3] compared to patients with the 4G allele but no FVL (reference group) or FVL irrespective of PAI-1 genotype (HR=1.8, 95 % CI=1.3-2.5). Compared to reference group, 5G allele irrespective of FVL was associated with lower risk of VTE recurrence only when compared with 4G allele together with FVL. In conclusion, FVL has a modifying effect on PAI-1 polymorphism in relation to risk of VTE recurrence. The role of PAI-1 polymorphism as a risk factor of recurrent VTE may be FVL dependent.

  14. Effects of pharmacological suppression of plasminogen activator inhibitor-1 in myocardial remodeling after ischemia reperfusion injury.

    PubMed

    Watanabe, Ryo; Nakajima, Takuya; Ogawa, Masahito; Suzuki, Jun-ichi; Muto, Susumu; Itai, Akiko; Hirata, Yasunobu; Nagai, Ryozo; Isobe, Mitsuaki

    2011-01-01

    Plasminogen activator inhibitor-1 (PAI-1) contributes to cardiac ventricular remodeling because migration of inflammatory cells and attenuation of extracellular matrix degradation are caused by plasmin and matrix metalloproteinase. However, the roles of PAI-1 in myocardial ischemia reperfusion (I/R) injury and the following inflammatory response have not yet been well elucidated. To clarify the role of PAI-1 in myocardial I/R injury, we used a specific PAI-1 inhibitor (IMD-1622) in a rat model. The left anterior descending coronary artery was ligated and reperfusion was performed by loosening the suture after 30 minutes of arterial occlusion. A single administration of IMD-1622 (20 mg/kg) or vehicle was given intraperitoneally and then the rats were sacrificed on day 1 or day 14 after I/R. Blood pressure, echocardiograms, histopathology, and molecular examination were performed. The examinations revealed that PAI-1 inhibitor showed limited effects on cardiac dysfunction and ventricular remodeling after I/R. We conclude that the pharmacological inhibition of PAI-1 may not affect ventricular remodeling after myocardial I/R injury.

  15. The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis Cleaves and inactivates plasminogen activator inhibitor type 1.

    PubMed

    Urano, T; Ihara, H; Umemura, K; Suzuki, Y; Oike, M; Akita, S; Tsukamoto, Y; Suzuki, I; Takada, A

    2001-07-01

    In this report, we demonstrate an interaction between subtilisin NAT (formerly designated BSP, or nattokinase), a profibrinolytic serine proteinase from Bacillus subtilis, and plasminogen activator inhibitor 1 (PAI-1). Subtilisin NAT was purified to homogeneity (molecular mass, 27.7 kDa) from a saline extract of B. subtilis (natto). Subtilisin NAT appeared to cleave active recombinant prokaryotic PAI-1 (rpPAI-1) into low molecular weight fragments. Matrix-assisted laser desorption/ionization in combination with time-of-flight mass spectroscopy and peptide sequence analysis revealed that rpPAI-1 was cleaved at its reactive site (P1-P1': Arg(346)-Met(347)). rpPAI-1 lost its specific activity after subtilisin NAT treatment in a dose-dependent manner (0.02-1.0 nm; half-maximal effect at approximately 0.1 nm). Subtilisin NAT dose dependently (0.06-1 nm) enhanced tissue-type plasminogen activator-induced fibrin clot lysis both in the absence of rpPAI-1 (48 +/- 1.4% at 1 nm) and especially in the presence of rpPAI-1 (78 +/- 2.0% at 1 nm). The enhancement observed in the absence of PAI-1 seems to be induced through direct fibrin dissolution by subtilisin NAT. The stronger enhancement by subtilisin NAT of rpPAI-1-enriched fibrin clot lysis seems to involve the cleavage and inactivation of active rpPAI-1. This mechanism is suggested to be important for subtilisin NAT to potentiate fibrinolysis.

  16. Adenoviral overexpression and small interfering RNA suppression demonstrate that plasminogen activator inhibitor-1 produces elevated collagen accumulation in normal and keloid fibroblasts.

    PubMed

    Tuan, Tai-Lan; Hwu, Paul; Ho, Wendy; Yiu, Peter; Chang, Richard; Wysocki, Annette; Benya, Paul D

    2008-11-01

    Keloids are tumor-like skin scars that grow as a result of the aberrant healing of skin injuries, with no effective treatment. We provide new evidence that both overexpression of plasminogen activator inhibitor-1 (PAI-1) and elevated collagen accumulation are intrinsic features of keloid fibroblasts and that these characteristics are causally linked. Using seven strains each of early passage normal and keloid fibroblasts, the keloid strains exhibited inherently elevated collagen accumulation and PAI-1 expression in serum-free, 0.1% ITS+ culture; larger increases in these parameters occurred when cells were cultured in 3% serum. To demonstrate a causal relationship between PAI-1 overexpression and collagen accumulation, normal fibroblasts were infected with PAI-1-expressing adenovirus. Such cells exhibited a two- to fourfold increase in the accumulation of newly synthesized collagen in a viral dose-dependent fashion in both monolayers and fibrin gel, provisional matrix-like cultures. Three different PAI-1-targeted small interfering RNAs, alone or in combination, produced greater than an 80% PAI-1 knockdown and reduced collagen accumulation in PAI-1-overexpressing normal or keloid fibroblasts. A vitronectin-binding mutant of PAI-1 was equipotent with wild-type PAI-1 in inducing collagen accumulation, whereas a complete protease inhibitor mutant retained approximately 50% activity. Thus, PAI-1 may use more than its protease inhibitory activity to control keloid collagen accumulation. PAI-1-targeted interventions, such as small interfering RNA and lentiviral short hairpin RNA-containing microRNA sequence suppression reported here, may have therapeutic utility in the prevention of keloid scarring.

  17. Plasminogen activator inhibitor 1, fibroblast apoptosis resistance, and aging-related susceptibility to lung fibrosis.

    PubMed

    Huang, Wen-Tan; Akhter, Hasina; Jiang, Chunsun; MacEwen, Mark; Ding, Qiang; Antony, Veena; Thannickal, Victor John; Liu, Rui-Ming

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF) is a fatal lung disorder with unknown cause and no effective treatment. The incidence of and mortality from IPF increase with age, suggesting that advanced age is a major risk factor for IPF. The mechanism underlying the increased susceptibility of the elderly to IPF, however, is unknown. In this study, we show for the first time that the protein level of plasminogen activator inhibitor 1 (PAI-1), a protease inhibitor which plays an essential role in the control of fibrinolysis, was significantly increased with age in mouse lung homogenate and lung fibroblasts. Upon bleomycin challenge, old mice experienced augmented PAI-1 induction and lung fibrosis as compared to young mice. Most interestingly, we show that fewer (myo)fibroblasts underwent apoptosis and more (myo)fibroblasts with increased level of PAI-1 accumulated in the lung of old than in young mice after bleomycin challenge. In vitro studies further demonstrate that fibroblasts isolated from lungs of old mice were resistant to H2O2 and tumor necrosis factor alpha-induced apoptosis and had augmented fibrotic responses to TGF-β1, compared to fibroblasts isolated from young mice. Inhibition of PAI-1 activity with a PAI-1 inhibitor, on the other hand, eliminated the aging-related apoptosis resistance and TGF-β1 sensitivity in isolated fibroblasts. Moreover, we show that knocking down PAI-1 in human lung fibroblasts with PAI-1 siRNA significantly increased their sensitivity to apoptosis and inhibited their responses to TGF-β1. Together, the results suggest that increased PAI-1 expression may underlie the aging-related sensitivity to lung fibrosis in part by protecting fibroblasts from apoptosis.

  18. Release of plasminogen activator inhibitor-1 from human astrocytes is regulated by intracellular ceramide.

    PubMed

    Kimura, M; Soeda, S; Oda, M; Ochiai, T; Kihara, T; Ono, N; Shimeno, H

    2000-12-15

    The present study underscores a regulatory role of intracellular ceramide in astrocytes for the release of an extracellular serine protease, tissue-type plasminogen activator (t-PA), and its inhibitor, plasminogen activator inhibitor-1 (PAI-1). Treatment of cultured human astrocytes with N-acetylsphingosine, a cell-permeable short-chain ceramide analogue or daunorubicin that could increase intracellular ceramide via activation of ceramide synthase or sphingomyelin hydrolysis increased the release of t-PA and conversely decreased the PAI-1 release. Interestingly, treatment of the astrocytes with tumor necrosis factor (TNF)-alpha also increased the intracellular ceramide levels but caused the elevation of PAI-1 release without altering the t-PA release. These data suggest that the generation of ceramide in astrocytes is linked at least with the regulation of PAI-1 release. We also demonstrate that the suppression of PAI-1 release with daunorubicin accelerates the cell death of neuronally differentiated PC12 cells and suggest an antiapoptotic role of PAI-1 in the nervous system.

  19. MiR-10a and miR-181c regulate collagen type I generation in hypertrophic scars by targeting PAI-1 and uPA.

    PubMed

    Li, Chao; Zhu, Hua-Yu; Bai, Wen-Dong; Su, Lin-Lin; Liu, Jia-Qi; Cai, Wei-Xia; Zhao, Bin; Gao, Jian-Xin; Han, Shi-Chao; Li, Jun; Hu, Da-Hai

    2015-01-30

    Urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) have been proposed to play key roles in extracellular matrix (ECM) deposition in hypertrophic scars (HS). Here, we found that in HS fibroblasts (HFs) miR-181c and miR-10a were differentially-expressed and targeted uPA and PAI-1, respectively. The production of Type 1 collagen (Col1) was inhibited by miR-181c knockdown or miR-10a overexpression in HFs, and this resulted in increased levels of metalloproteinase 1 (MMP1). These results suggest that the miR-181c-uPA and miR-10a-PAI-1 regulatory pathways have an integral role in HS pathogenesis.

  20. Cylindromatosis (CYLD) inhibits Streptococcus pneumonia-induced plasminogen activator inhibitor-1 expression via interacting with TRAF-6.

    PubMed

    Wu, Jing; Zhao, Jing; Yu, Jie; Zhang, Wenhong; Huang, Yuxian

    2015-08-01

    Streptococcus pneumoniae (S. p) remains one of the foremost causes of community-acquired pneumonia. Recent studies have shown that S. p lung infection is associated with plasminogen activator inhibitor-1 (PAI-1) expression, which inhibits acute lung injury. Such effects by S. p were negatively regulated by cylindromatosis (CYLD). The current study explored the underlying mechanisms. We showed that S. p-induced PAI-1 expression requires tumor necrosis factor receptor-associated factor 6 (TRAF-6) signaling. Si-RNA-mediated knockdown of TRAF-6 remarkably inhibited S. p-induced PAI-1 expression. Reversely, over-expression of wild type (wt-) TRAF-6 further potentiated PAI-1 expression in S. p-treated cells. We provided evidences to support that CYLD-mediated anti-PAI-1 activity might be through direct regulation of TRAF-6. Our results from co-immunoprecipitation (co-IP) and confocal microscopy assays confirmed a direct association between the CYLD and TRAF-6 in A549 cells. Over-expression of wt-CYLD remarkably inhibited TRAF-6 ubiquitination and subsequent PAI-1 expression. Introducing a mutated CYLD, on the other hand, enhanced TRAF-6 ubiquitination and PAI-1 expression. Together, these results indicate that TRAF-6 mediates S. p-induced PAI-1 expression, and CYLD inhibits PAI-1 expression probably through deubiquitinating TRAF-6. The current study provided molecular insights of CYLD-mediated activities in S. p-induced PAI-1 expression and possible acute lung injury.

  1. p53- and PAI-1-mediated induction of C-X-C chemokines and CXCR2: importance in pulmonary inflammation due to cigarette smoke exposure.

    PubMed

    Tiwari, Nivedita; Marudamuthu, Amarnath S; Tsukasaki, Yoshikazu; Ikebe, Mitsuo; Fu, Jian; Shetty, Sreerama

    2016-03-15

    We previously demonstrated that tumor suppressor protein p53 augments plasminogen activator inhibitor-1 (PAI-1) expression in alveolar epithelial cells (AECs) during chronic cigarette smoke (CS) exposure-induced lung injury. Chronic lung inflammation with elevated p53 and PAI-1 expression in AECs and increased susceptibility to and exacerbation of respiratory infections are all associated with chronic obstructive pulmonary disease (COPD). We recently demonstrated that preventing p53 from binding to the endogenous PAI-1 mRNA in AECs by either suppressing p53 expression or blockading p53 interactions with the PAI-1 mRNA mitigates apoptosis and lung injury. Within this context, we now show increased expression of the C-X-C chemokines (CXCL1 and CXCL2) and their receptor CXCR2, and the intercellular cellular adhesion molecule-1 (ICAM-1), in the lung tissues of patients with COPD. We also found a similar increase in lung tissues and AECs from wild-type (WT) mice exposed to passive CS for 20 wk and in primary AECs treated with CS extract in vitro. Interestingly, passive CS exposure of mice lacking either p53 or PAI-1 expression resisted an increase in CXCL1, CXCL2, CXCR2, and ICAM-1. Furthermore, inhibition of p53-mediated induction of PAI-1 expression by treatment of WT mice exposed to passive CS with caveolin-1 scaffolding domain peptide reduced CXCL1, CXCL2, and CXCR2 levels and lung inflammation. Our study reveals that p53-mediated induction of PAI-1 expression due to chronic CS exposure exacerbates lung inflammation through elaboration of CXCL1, CXCL2, and CXCR2. We further provide evidence that targeting this pathway mitigates lung injury associated with chronic CS exposure.

  2. Mechanisms of TGF-beta1-induced intimal growth: plasminogen-independent activities of plasminogen activator inhibitor-1 and heterogeneous origin of intimal cells.

    PubMed

    Otsuka, Goro; Stempien-Otero, April; Frutkin, Andrew D; Dichek, David A

    2007-05-11

    Transforming growth factor (TGF)-beta(1) is a potent stimulator of intimal growth. We showed previously that TGF-beta(1) stimulates intimal growth through early upregulation of plasminogen activator inhibitor-1 (PAI-1) and, subsequently, PAI-1-dependent increases in cell migration and matrix accumulation. We also showed that PAI-1 negatively regulates TGF-beta(1) expression in the artery wall. Here we use plasminogen-deficient mice to test whether TGF-beta(1)-stimulated, PAI-1-dependent intimal growth and PAI-1 suppression of TGF-beta(1) expression are mediated through inhibition of plasminogen activation by PAI-1. We also use lineage tracing to investigate the origin of cells in TGF-beta(1)-induced intimas. Surprisingly, both TGF-beta(1)-induced, PAI-1-dependent intimal growth and PAI-1 suppression of TGF-beta(1) expression are independent of plasminogen. Moreover, approximately 50% of cells that migrate into the intima of TGF-beta(1)-overexpressing arteries carry a smooth muscle lineage marker, <1% carry a bone marrow lineage marker, and the remaining cells carry neither marker. Therefore, PAI-1 stimulates intimal growth and suppresses TGF-beta(1) expression through activities other than inhibition of plasminogen activation. In addition, contrary to widely held models, our results do not support a role for plasmin (or thrombospondin) in TGF-beta(1) activation in the artery wall. Further identification of the molecular targets through which PAI-1 stimulates intimal formation and suppresses TGF-beta(1) expression in the artery wall may reveal new approaches for inhibiting intimal formation. Our studies also discount bone marrow as an important source from which TGF-beta(1) recruits intimal cells and suggest instead that TGF-beta(1) induces substantial cell migration either from the adventitia or from an extravascular, but nonhematopoietic source.

  3. Elevated circulating PAI-1 levels are related to lung function decline, systemic inflammation, and small airway obstruction in chronic obstructive pulmonary disease

    PubMed Central

    Wang, Hao; Yang, Ting; Li, Diandian; Wu, Yanqiu; Zhang, Xue; Pang, Caishuang; Zhang, Junlong; Ying, Binwu; Wang, Tao; Wen, Fuqiang

    2016-01-01

    Background Plasminogen activator inhibitor-1 (PAI-1) and soluble urokinase-type plasminogen activator receptor (suPAR) participate in inflammation and tissue remolding in various diseases, but their roles in chronic obstructive pulmonary disease (COPD) are not yet clear. This study aimed to investigate if PAI-1 and suPAR were involved in systemic inflammation and small airway obstruction (SAO) in COPD. Methods Demographic and clinical characteristics, spirometry examination, and blood samples were obtained from 84 COPD patients and 51 healthy volunteers. Serum concentrations of PAI-1, suPAR, tissue inhibitor of metalloproteinase-1 (TIMP-1), Matrix metalloproteinase-9 (MMP-9), and C-reactive protein (CRP) were detected with Magnetic Luminex Screening Assay. Differences between groups were statistically analyzed using one-way analysis of variance or chi-square test. Pearson’s partial correlation test (adjusted for age, sex, body mass index, cigarette status, and passive smoke exposure) and multivariable linear analysis were used to explore the relationships between circulating PAI-1 and indicators of COPD. Results First, we found that serum PAI-1 levels but not suPAR levels were significantly increased in COPD patients compared with healthy volunteers (125.56±51.74 ng/mL versus 102.98±36.62 ng/mL, P=0.007). Then, the correlation analysis showed that circulating PAI-1 was inversely correlated with pulmonary function parameters including the ratio of forced expiratory volume in 1 second to forced vital capacity (FEV1/FVC), FEV1/Pre (justified r=−0.308, P<0.001; justified r=−0.295, P=0.001, respectively) and SAO indicators such as FEV3/FVC, MMEF25–75/Pre (justified r=−0.289, P=0.001; justified r=−0.273, P=0.002, respectively), but positively related to the inflammatory marker CRP (justified r=0.351, P<0.001), the small airway remolding biomarker TIMP-1, and MMP-9 (justified r=0.498, P<0.001; justified r=0.267, P=0.002, respectively). Besides, multivariable

  4. Plasminogen activator inhibitor-1 regulates LPS-induced TLR4/MD-2 pathway activation and inflammation in alveolar macrophages.

    PubMed

    Ren, Weiying; Wang, Zhonghui; Hua, Feng; Zhu, Lei

    2015-02-01

    Toll-like receptor 4 (TLR4) and myeloid differentiation protein 2 (MD-2) are the main lipopolysaccharide (LPS) binding receptors that respond to inflammatory stimuli and mediate NF-kappa B (NF-κB) signaling pathway in macrophages. We have previously shown that plasminogen activator inhibitor-1 (PAI-1) deletion increased lung injury induced by intratracheal instillation of LPS through downregulation of TLR4 negative regulators. However, the mechanisms by which PAI-1 regulates lung inflammation are largely unknown. The aim of this study is to assess the relationship between PAI-1 and TLR4 signaling pathways in LPS-induced NR8383 cells inflammatory reaction. The results showed that the levels of PAI-1, TNF-α, and IL-1β protein were increased remarkably in NR8383 cell supernatants after LPS stimulation. PAI-1 gene knockdown reduced TNF-α and IL-1β levels in cell supernatants and inhibited the NF-κB p65 protein expression in NR8383 cells. The upregulated mRNA and protein expressions of TLR4, MD-2, and myeloid differentiation protein (MyD88) induced by LPS were attenuated after PAI-1 gene knockdown. Conversely, overexpression of PAI-1 in NR8383 cells not only resulted in additional mRNA and protein production of PAI-1, TLR4, MD-2, and MyD88, it also aggravated the inflammatory response induced by LPS. In conclusion, PAI-1 contributes to the regulation of LPS-induced inflammatory response in NR8383 cells, likely by affecting the TLR4-MD-2/NF-κB signaling transduction pathway.

  5. Rosiglitazone prevents advanced glycation end products-induced renal toxicity likely through suppression of plasminogen activator inhibitor-1.

    PubMed

    Yu, Xiaoyan; Li, Cai; Li, Xiaokun; Cai, Lu

    2007-04-01

    In the development of diabetic nephropathy, advanced glycation end products (AGEs) play a causative role via induction of extracellular matrix (ECM) accumulation. Plasminogen activator inhibitor-1 (PAI-1), as a major inhibitor of plasminogen activator that plays an important role in degrading ECM, was found to significantly increase in renal fibrotic diseases. Activation of peroxisome proliferator-activated receptor (PPAR)-gamma prevented diabetic nephropathy. The present study, therefore, was to define whether or not AGE-induced renal ECM accumulation and renal dysfunction are mediated by upregulation of PAI-1 expression and whether or not PPAR-gamma agonist can attenuate these AGE effects via suppressing PAI-1 expression. Rats were given AGEs alone by iv injection at 100 mg/kg daily with or without oral supplementation of PPAR-gamma agonist rosiglitazone (RGZ) at 2 mg/kg daily for 6 weeks. Results showed that AGEs induced a renal ECM accumulation, as shown by increases in periodic acid-Schiff-positive materials, fibronectin, and type IV collagen (Col IV) contents in glomeruli, and a mild renal dysfunction, as shown by an increase in urinary proteins. AGEs also caused an increase in PAI-1 expression and a decrease in plasminogen activator bioactivity in the kidney. Treatment with RGZ significantly ameliorated AGE-induced renal ECM accumulation, proteinuria, and PAI-1 upregulation. Direct exposure of rat mesangial cells to AGEs in vitro induced increases in fibronectin and Col IV syntheses along with an increase in PAI-1 expression, effects significantly attenuated by RGZ. Preincubation of PAI-1 antibody to AGE-treated mesangial cells completely prevented AGE-induced fibronectin and Col IV production. These results suggest that upregulation of PAI-1 expression plays a critical role in AGE-induced renal ECM accumulation. Renal protection of RGZ from AGEs may be associated with the suppression of PAI-1 expression through PPAR-dependent and independent mechanisms.

  6. Atrial natriuretic peptide inhibits the expression of tissue factor and plasminogen activator inhibitor 1 induced by angiotensin II in cultured rat aortic endothelial cells.

    PubMed

    Yoshizumi, M; Tsuji, H; Nishimura, H; Kasahara, T; Sugano, T; Masuda, H; Nakagawa, K; Nakahara, Y; Kitamura, H; Yamada, K; Yoneda, M; Sawada, S; Nakagawa, M

    1998-03-01

    The pharmacological characteristics of atrial natriuretic peptide (ANP), such as natriuresis, vasodilation, or suppression of smooth muscle cell proliferation, are well investigated. However, this is the first study to report its role on blood coagulation and fibrinolysis mediated by vascular endothelial cells. In this study, the effects of ANP on the enhanced expression of tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) by angiotensin II (Ang II) in cultured rat aortic endothelial cells (RAECs) were examined. The expressions of TF and PAI-1 mRNA were detected by northern blotting methods. The activities of TF on the surface of RAECs and PAI-1 in the culture media were measured by chromogenic assay. ANP suppressed mRNA expressions of TF and PAI-1 induced by Ang II in a concentration-dependent manner. This suppression was accompanied by the decreased activities of TF and PAI-1.

  7. 1,25-Dihydroxyvitamin D3 Suppresses Inflammation-Induced Expression of Plasminogen Activator Inhibitor-1 by Blocking Nuclear Factor-κB Activation

    PubMed Central

    Chen, Yunzi; Kong, Juan; Sun, Tao; Li, George; Szeto, Frances L.; Liu, Weicheng; Deb, Dilip K.; Wang, Youli; Zhao, Qun; Thadhani, Ravi; Li, Yan Chun

    2011-01-01

    Plasminogen activator inhibitor (PAI)-1 is a major fibrinolytic inhibitor. High PAI-1 is associated with increased renal and cardiovascular disease risk. Previous studies demonstrated PAI-1 down-regulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), but the molecular mechanism remains unknown. Here we show that exposure of mouse embryonic fibroblasts to TNFα or LPS led to a marked induction of PAI-1, which was blunted by 1,25(OH)2D3, NF-κB inhibitor or p65 siRNA, suggesting the involvement of NF-κB in 1,25(OH)2D3-induced repression. In mouse Pai-1 promoter a putative cis-κB element was identified at −299. EMSA and ChIP assays showed that TNF-α increased p65/p50 binding to this κB site, which was disrupted by 1,25(OH)2D3. Luciferase reporter assays showed that PAI-1 promoter activity was induced by TNFα or LPS, and the induction was blocked by 1,25(OH)2D3. Mutation of the κB site blunted TNFα, LPS or 1,25(OH)2D3 effects. 1,25(OH)2D3 blocked IκBα degradation and arrested p50/p65 nuclear translocation. In mice LPS stimulated PAI-1 expression in the heart and macrophages, and the stimulation was blunted by pre-treatment with a vitamin D analog. Together these data demonstrate that 1,25(OH)2D3 down-regulates PAI-1 by blocking NF-κB activation. Inhibition of PAI-1 production may contribute to the reno- and cardio-protective effects of vitamin D. PMID:21176770

  8. Elevated Cytokines, Thrombin and PAI-1 in Severe HCPS Patients Due to Sin Nombre Virus

    PubMed Central

    Bondu, Virginie; Schrader, Ron; Gawinowicz, Mary Ann; McGuire, Paul; Lawrence, Daniel A.; Hjelle, Brian; Buranda, Tione

    2015-01-01

    Sin Nombre Hantavirus (SNV, Bunyaviridae Hantavirus) is a Category A pathogen that causes Hantavirus Cardiopulmonary Syndrome (HCPS) with case fatality ratios generally ranging from 30% to 50%. HCPS is characterized by vascular leakage due to dysregulation of the endothelial barrier function. The loss of vascular integrity results in non-cardiogenic pulmonary edema, shock, multi-organ failure and death. Using Electric Cell-substrate Impedance Sensing (ECIS) measurements, we found that plasma samples drawn from University of New Mexico Hospital patients with serologically-confirmed HCPS, induce loss of cell-cell adhesion in confluent epithelial and endothelial cell monolayers grown in ECIS cultureware. We show that the loss of cell-cell adhesion is sensitive to both thrombin and plasmin inhibitors in mild cases, and to thrombin only inhibition in severe cases, suggesting an increasing prothrombotic state with disease severity. A proteomic profile (2D gel electrophoresis and mass spectrometry) of HCPS plasma samples in our cohort revealed robust antifibrinolytic activity among terminal case patients. The prothrombotic activity is highlighted by acute ≥30 to >100 fold increases in active plasminogen activator inhibitor (PAI-1) which, preceded death of the subjects within 48 h. Taken together, this suggests that PAI-1 might be a response to the severe pathology as it is expected to reduce plasmin activity and possibly thrombin activity in the terminal patients. PMID:25674766

  9. UPA and PAI-1 analysis from fixed tissues - new perspectives for a known set of predictive markers.

    PubMed

    Malinowsky, K; Böllner, C; Hipp, S; Berg, D; Schmitt, M; Becker, K F

    2010-01-01

    The urokinase-type plasminogen activator (uPA) and its main inhibitor PAI-1 play key roles in tumor-associated processes such as the degradation of the extracellular matrix (ECM), tissue remodeling, cell adhesion and migration. Elevated expression of both molecules is known to correlate with negative outcomes in node negative breast cancer. To date, these molecules are the only prognostic markers to have reached the highest level of evidence (LOE I) in multi-centered clinical trials for prognosis of node negative breast cancer. Unfortunately, the clinical utility of these molecules as markers is limited by the use of enzyme-linked immunoassay (ELISA) tests for their detection. The ELISA relies on the use of fresh or frozen tissue, which are rarely available in routine clinical settings. In this review article, we provide an overview of the clinical relevance of uPA and PAI-1 and present alternative methods for their detection. Common uPA and PAI-1 detection methods discussed in literature include RT-PCR-based assays and classical immunohistochemistry approaches. In recent years, attempts have been made to isolate and analyze proteins of formalin fixed, paraffin embedded (FFPE) tissues. These new methods are of special interest because up to now neither RT-PCR nor immunohistochemistry are recommended for the detection of uPA and PAI-1. Here, we present an approach for the analysis of uPA and PAI-1 directly from FFPE tissues that may eventually overcome the limitations of current assays and make the use of both markers widely available for routine prognosis and therapy decisions for breast cancer patients.

  10. Inhibition of Plasminogen Activator Inhibitor-1 Attenuates Transforming Growth Factor-β-Dependent Epithelial Mesenchymal Transition and Differentiation of Fibroblasts to Myofibroblasts

    PubMed Central

    Omori, Keitaro; Hattori, Noboru; Senoo, Tadashi; Takayama, Yusuke; Masuda, Takeshi; Nakashima, Taku; Iwamoto, Hiroshi; Fujitaka, Kazunori; Hamada, Hironobu; Kohno, Nobuoki

    2016-01-01

    Transforming growth factor-β (TGF-β) is central during the pathogenesis of pulmonary fibrosis, in which the plasminogen activator inhibitor-1 (PAI-1) also has an established role. TGF-β is also known to be the strongest inducer of PAI-1. To investigate the link between PAI-1 and TGF-β in fibrotic processes, we evaluated the effect of SK-216, a PAI-1-specific inhibitor, in TGF-β-dependent epithelial-mesenchymal transition (EMT) and fibroblast to myofibroblast differentiation. In human alveolar epithelial A549 cells, treatment with TGF-β induced EMT, whereas co-treatment with SK-216 attenuated the occurrence of EMT. The inhibition of TGF-β-induced EMT by SK-216 was also confirmed in the experiment using murine epithelial LA-4 cells. Blocking EMT by SK-216 inhibited TGF-β-induced endogenous production of PAI-1 and TGF-β in A549 cells as well. These effects of SK-216 were not likely mediated by suppressing either Smad or ERK pathways. Using human lung fibroblast MRC-5 cells, we demonstrated that SK-216 inhibited TGF-β-dependent differentiation of fibroblasts to myofibroblasts. We also observed this inhibition by SK-216 in human primary lung fibroblasts. Following these in vitro results, we tested oral administration of SK-216 into mice injected intratracheally with bleomycin. We found that SK-216 reduced the degree of bleomycin-induced pulmonary fibrosis in mice. Although the precise mechanisms underlying the link between TGF-β and PAI-1 regarding fibrotic process were not determined, PAI-1 seems to act as a potent downstream effector on the pro-fibrotic property of TGF-β. In addition, inhibition of PAI-1 activity by a PAI-1 inhibitor exerts an antifibrotic effect even in vivo. These data suggest that targeting PAI-1 as a downstream effector of TGF-β could be a promising therapeutic strategy for pulmonary fibrosis. PMID:26859294

  11. Inhibition of Plasminogen Activator Inhibitor-1 Attenuates Transforming Growth Factor-β-Dependent Epithelial Mesenchymal Transition and Differentiation of Fibroblasts to Myofibroblasts.

    PubMed

    Omori, Keitaro; Hattori, Noboru; Senoo, Tadashi; Takayama, Yusuke; Masuda, Takeshi; Nakashima, Taku; Iwamoto, Hiroshi; Fujitaka, Kazunori; Hamada, Hironobu; Kohno, Nobuoki

    2016-01-01

    Transforming growth factor-β (TGF-β) is central during the pathogenesis of pulmonary fibrosis, in which the plasminogen activator inhibitor-1 (PAI-1) also has an established role. TGF-β is also known to be the strongest inducer of PAI-1. To investigate the link between PAI-1 and TGF-β in fibrotic processes, we evaluated the effect of SK-216, a PAI-1-specific inhibitor, in TGF-β-dependent epithelial-mesenchymal transition (EMT) and fibroblast to myofibroblast differentiation. In human alveolar epithelial A549 cells, treatment with TGF-β induced EMT, whereas co-treatment with SK-216 attenuated the occurrence of EMT. The inhibition of TGF-β-induced EMT by SK-216 was also confirmed in the experiment using murine epithelial LA-4 cells. Blocking EMT by SK-216 inhibited TGF-β-induced endogenous production of PAI-1 and TGF-β in A549 cells as well. These effects of SK-216 were not likely mediated by suppressing either Smad or ERK pathways. Using human lung fibroblast MRC-5 cells, we demonstrated that SK-216 inhibited TGF-β-dependent differentiation of fibroblasts to myofibroblasts. We also observed this inhibition by SK-216 in human primary lung fibroblasts. Following these in vitro results, we tested oral administration of SK-216 into mice injected intratracheally with bleomycin. We found that SK-216 reduced the degree of bleomycin-induced pulmonary fibrosis in mice. Although the precise mechanisms underlying the link between TGF-β and PAI-1 regarding fibrotic process were not determined, PAI-1 seems to act as a potent downstream effector on the pro-fibrotic property of TGF-β. In addition, inhibition of PAI-1 activity by a PAI-1 inhibitor exerts an antifibrotic effect even in vivo. These data suggest that targeting PAI-1 as a downstream effector of TGF-β could be a promising therapeutic strategy for pulmonary fibrosis.

  12. Plasminogen activator inhibitor-1 suppresses profibrotic responses in fibroblasts from fibrotic lungs.

    PubMed

    Marudamuthu, Amarnath S; Shetty, Shwetha K; Bhandary, Yashodhar P; Karandashova, Sophia; Thompson, Michael; Sathish, Venkatachalem; Florova, Galina; Hogan, Taryn B; Pabelick, Christina M; Prakash, Y S; Tsukasaki, Yoshikazu; Fu, Jian; Ikebe, Mitsuo; Idell, Steven; Shetty, Sreerama

    2015-04-10

    Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive interstitial scarification. A hallmark morphological lesion is the accumulation of myofibroblasts or fibrotic lung fibroblasts (FL-fibroblasts) in areas called fibroblastic foci. We previously demonstrated that the expression of both urokinase-type plasminogen activator (uPA) and the uPA receptor are elevated in FL-fibroblasts from the lungs of patients with IPF. FL-fibroblasts isolated from human IPF lungs and from mice with bleomycin-induced pulmonary fibrosis showed an increased rate of proliferation compared with normal lung fibroblasts (NL-fibroblasts) derived from histologically "normal" lung. Basal expression of plasminogen activator inhibitor-1 (PAI-1) in human and murine FL-fibroblasts was reduced, whereas collagen-I and α-smooth muscle actin were markedly elevated. Conversely, alveolar type II epithelial cells surrounding the fibrotic foci in situ, as well as those isolated from IPF lungs, showed increased activation of caspase-3 and PAI-1 with a parallel reduction in uPA expression. Transduction of an adenovirus PAI-1 cDNA construct (Ad-PAI-1) suppressed expression of uPA and collagen-I and attenuated proliferation in FL-fibroblasts. On the contrary, inhibition of basal PAI-1 in NL-fibroblasts increased collagen-I and α-smooth muscle actin. Fibroblasts isolated from PAI-1-deficient mice without lung injury also showed increased collagen-I and uPA. These changes were associated with increased Akt/phosphatase and tensin homolog proliferation/survival signals in FL-fibroblasts, which were reversed by transduction with Ad-PAI-1. This study defines a new role of PAI-1 in the control of fibroblast activation and expansion and its role in the pathogenesis of fibrosing lung disease and, in particular, IPF.

  13. A Mechanism for Assembly of Complexes of Vitronectin and Plasminogen Activator Inhibitor-1 from Sedimmentation Velocity Analysis*

    PubMed Central

    Minor, Kenneth H.; Schar, Christine R.; Blouse, Grant E.; Shore, Joseph D.; Lawrence, Daniel A.; Schuck, Peter; Peterson, Cynthia B.

    2005-01-01

    Plasminogen activator inhibitor-1 (PAI-1) and vitronectin are cofactors involved in pathological conditions such as injury, inflammation, and cancer, during which local levels of PAI-1 are increased and the active serpin forms complexes with vitronectin. These complexes become deposited into surrounding tissue matrices, where they regulate cell adhesion and peri-cellular proteolysis. The mechanism for their co-localization has not been elucidated. We hypothesize that PAI-1-vitronectin complexes form in a stepwise and concentration-dependent fashion via 1:1 and 2:1 intermediates, with the 2:1 complex serving a key role in assembly of higher order complexes. To test this hypothesis, sedimentation velocity experiments in the analytical ultracentrifuge were performed to identify different PAI-1-vitronectin complexes. Analysis of sedimentation data invoked a novel multisignal method to discern the stoichiometry of the two proteins in the higher-order complexes formed (Balbo, A., Minor, K. H., Velikovsky, C. A., Mariuzza, R. A., Peterson, C. B., and Schuck, P. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 81—86). Our results demonstrate that PAI-1 and vitronectin assemble into higher order forms via a pathway that is triggered upon saturation of the two PAI-1-binding sites of vitronectin to form the 2:1 complex. This 2:1 PAI-1-vitronectin complex, with a sedimentation coefficient of 6.5 S, is the key intermediate for the assembly of higher order complexes. PMID:15905170

  14. Plasminogen activator inhibitor 1 and insulin levels in various insulin resistance states.

    PubMed

    Scelles, V; Raccah, D; Alessi, M C; Vialle, J M; Juhan-Vague, I; Vague, P

    1992-01-01

    Among obese insulin resistant subjects plasminogen activator inhibitor 1 (PAI 1) levels are closely associated with fasting insulin levels in cross sectional as well as intervention studies. Insulin concentration by itself does not seem to modulate PAI 1 levels at least in acute conditions. PAI 1 levels could be more directly related to the insulin resistant state than to hyperinsulinaemia. To elucidate further this phenomenon we compared insulin, triglyceride and PAI 1 levels in twenty control subjects and in three groups of patients presenting insulin resistance 14 obese subjects, 6 patients with Cushing disease and 7 with acromegaly. None of the tested subjects was diabetic. Fasting insulin levels were elevated in obese (21.4 +/- 8.0) hypercortisolic (20.3 +/- 11.0) and acromegalic patients (16.1 +/- 5.0) compared to controls (9.2 +/- 3.0 microU/ml, m +/- SD). PAI activity and PAI 1 antigen levels were elevated in the obese group only (34.3 +/- 13.0 for PAI 1 activity) and not in the others: 10.2 +/- 10.0, 7.0 +/- 4.6 I U/l for hypercortisolic and acromegalic patients respectively (normal controls 9.7 +/- 5.4). Triglyceride levels were also elevated among obese subjects 2.2 +/- 1.3 vs 1.1 +/- 0.4 mM/l in the controls; they were slightly higher than normal but not significantly in the hypercortisolic (1.5 +/- 0.6) and acromegalic (1.43 +/- 0.6 mM/l) patients. The mechanism of insulin resistance is different in the three conditions studied here. This may explain why elevated PAI 1 concentration are restricted to the common form of insulin resistance as seen in obese subjects. Therefore insulin resistant state per se is not associated with elevated PAI 1 levels.

  15. Plasminogen activator inhibitor type 1 gene is located at region q21. 3-q22 of chromosome 7 and genetically linked with cystic fibrosis

    SciTech Connect

    Klinger, K.W.; Winqvist, R.; Riccio, A.; Andreasen, P.A.; Sartorio, R.; Nielsen, L.S.; Stuart, N.; Stanislovitis, P.; Watkins, P.; Douglas, R.

    1987-12-01

    The regional chromosomal location of the human gene for plasminogen activator inhibitor type 1 (PAI1) was determined by three independent methods of gene mapping. PAI1 was localized first to 7cen-q32 and then to 7q21.3-q22 by Southern blot hybridization analysis of a panel of human and mouse somatic cell hybrids with a PAI1 cDNA probe and in situ hybridization, respectively. The authors frequent HindIII restriction fragment length polymorphism (RFLP) of the PAI1 gene with an information content of 0.369. In family studies using this polymorphism, genetic linkage was found between PAI1 and the loci for erythropoietin (EPO), paraoxonase (PON), the met protooncogene (MET), and cystic fibrosis (CF), all previously assigned to the middle part of the long arm of chromosome 7. The linkage with EPO was closest with an estimated genetic distance of 3 centimorgans, whereas that to CF was 20 centimorgans. A three-point genetic linkage analysis and data from previous studies showed that the most likely order of these loci is EPO, PAI1, PON, (MET, CF), with PAI1 being located centromeric to CF. The PAI1 RFLP may prove to be valuable in ordering genetic markers in the CF-linkage group and may also be valuable in genetic analysis of plasminogen activation-related diseases, such as certain thromboembolic disorders and cancer.

  16. Epistatic effects of polymorphisms in genes from the renin-angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels.

    PubMed

    Asselbergs, Folkert W; Williams, Scott M; Hebert, Patricia R; Coffey, Christopher S; Hillege, Hans L; Navis, Gerjan; Vaughan, Douglas E; van Gilst, Wiek H; Moore, Jason H

    2007-03-01

    Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) directly influence thrombus formation and degradation and thereby risk for arterial thrombosis. Activation of the renin-angiotensin system has been linked to the production of PAI-1 expression via the angiotensin II type 1 receptor (AT1R). In addition, bradykinin can induce the release of t-PA through a B2 receptor mechanism. In the present study, we aimed to investigate the epistatic effects of polymorphisms in genes from the renin-angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels in a large population-based sample (n=2527). We demonstrated a strong significant interaction within genetic variations of the bradykinin B2 gene (P=0.002) and between ACE and bradykinin B2 (p=0.003) polymorphisms on t-PA levels in females. In males, polymorphisms in the bradykinin B2 and AT1R gene showed the most strong effect on t-PA levels (P=0.006). In both females and males, the bradykinin B2 gene interacted with AT1R gene on plasma PAI-1 levels (P=0.026 and P=0.039, respectively). In addition, the current study found a borderline significant interaction between PAI 4G5G and ACE I/D on plasma t-PA and PAI-1 levels. These results support the idea that the interplay between the renin-angiotensin, bradykinin, and fibrinolytic systems might play an important role in t-PA and PAI-1 biology.

  17. Effect of ascorbate on plasminogen activator inhibitor-1 expression and release from platelets and endothelial cells in an in-vitro model of sepsis.

    PubMed

    Swarbreck, Scott B; Secor, Dan; Ellis, Christopher G; Sharpe, Michael D; Wilson, John X; Tyml, Karel

    2015-06-01

    The microcirculation during sepsis fails due to capillary plugging involving microthrombosis. We demonstrated that intravenous injection of ascorbate reduces this plugging, but the mechanism of this beneficial effect remains unclear. We hypothesize that ascorbate inhibits the release of the antifibrinolytic plasminogen activator inhibitor-1 (PAI-1) from endothelial cells and platelets during sepsis. Microvascular endothelial cells and platelets were isolated from mice. Cells were cultured and stimulated with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα), or thrombin (agents of sepsis), with/without ascorbate for 1-24 h. PAI-1 mRNA was determined by quantitative PCR. PAI-1 protein release into the culture medium was measured by ELISA. In platelets, PAI-1 release was measured after LPS, TNFα, or thrombin stimulation, with/without ascorbate. In endothelial cells, LPS and TNFα increased PAI-1 mRNA after 6-24 h, but no increase in PAI-1 release was observed; ascorbate did not affect these responses. In platelets, thrombin, but not LPS or TNFα, increased PAI-1 release; ascorbate inhibited this increase at low extracellular pH. In unstimulated endothelial cells and platelets, PAI-1 is released into the extracellular space. Thrombin increases this release from platelets; ascorbate inhibits it pH-dependently. The data suggest that ascorbate promotes fibrinolysis in the microvasculature under acidotic conditions in sepsis. PMID:25730478

  18. Effect of ascorbate on plasminogen activator inhibitor-1 expression and release from platelets and endothelial cells in an in-vitro model of sepsis.

    PubMed

    Swarbreck, Scott B; Secor, Dan; Ellis, Christopher G; Sharpe, Michael D; Wilson, John X; Tyml, Karel

    2015-06-01

    The microcirculation during sepsis fails due to capillary plugging involving microthrombosis. We demonstrated that intravenous injection of ascorbate reduces this plugging, but the mechanism of this beneficial effect remains unclear. We hypothesize that ascorbate inhibits the release of the antifibrinolytic plasminogen activator inhibitor-1 (PAI-1) from endothelial cells and platelets during sepsis. Microvascular endothelial cells and platelets were isolated from mice. Cells were cultured and stimulated with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα), or thrombin (agents of sepsis), with/without ascorbate for 1-24 h. PAI-1 mRNA was determined by quantitative PCR. PAI-1 protein release into the culture medium was measured by ELISA. In platelets, PAI-1 release was measured after LPS, TNFα, or thrombin stimulation, with/without ascorbate. In endothelial cells, LPS and TNFα increased PAI-1 mRNA after 6-24 h, but no increase in PAI-1 release was observed; ascorbate did not affect these responses. In platelets, thrombin, but not LPS or TNFα, increased PAI-1 release; ascorbate inhibited this increase at low extracellular pH. In unstimulated endothelial cells and platelets, PAI-1 is released into the extracellular space. Thrombin increases this release from platelets; ascorbate inhibits it pH-dependently. The data suggest that ascorbate promotes fibrinolysis in the microvasculature under acidotic conditions in sepsis.

  19. Plasminogen activator inhibitor-1 as an early potential diagnostic marker for Alzheimer's disease.

    PubMed

    Oh, Jaeho; Lee, Hye-Ja; Song, Ji-Hyun; Park, Sang Ick; Kim, Hyunyoung

    2014-12-01

    Alzheimer's disease (AD) is the most common cause of dementia in individuals over 65 years old. However, to date, no useful early diagnostic markers for AD have been discovered. We examined the utility of plasminogen activator inhibitor-1 (PAI-1) as a potential biomarker for AD in subjects with mild cognitive impairment (MCI) or AD, as well as in nondemented healthy controls. Plasma PAI-1 levels were measured by enzyme-linked immunosorbent assays (ELISAs) in samples collected from 76 patients with MCI, 74 patients with AD, and 76 healthy controls. Our results show that plasma PAI-1 levels gradually increased as dementia progressed. The mean levels of plasma PAI-1 in patients with MCI and AD patients were significantly higher than those of in healthy controls. Consistently, neuropsychological examination (e.g., MMSE, CDR) also demonstrated significant correlations between the plasma PAI-1 levels and cognitive function. In conclusion, the level of plasma PAI-1 is a potential biomarker for the early detection and diagnosis of AD.

  20. Therapeutic potential of an orally effective small molecule inhibitor of plasminogen activator inhibitor for asthma.

    PubMed

    Liu, Rui-Ming; Eldridge, Stephanie; Watanabe, Nobuo; Deshane, Jessy; Kuo, Hui-Chien; Jiang, Chunsun; Wang, Yong; Liu, Gang; Schwiebert, Lisa; Miyata, Toshio; Thannickal, Victor J

    2016-02-15

    Asthma is one of the most common respiratory diseases. Although progress has been made in our understanding of airway pathology and many drugs are available to relieve asthma symptoms, there is no cure for chronic asthma. Plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of tissue-type and urokinase-type plasminogen activators, has pleiotropic functions besides suppression of fibrinolysis. In this study, we show that administration of TM5275, an orally effective small-molecule PAI-1 inhibitor, 25 days after ovalbumin (OVA) sensitization-challenge, significantly ameliorated airway hyperresponsiveness in an OVA-induced chronic asthma model. Furthermore, we show that TM5275 administration significantly attenuated OVA-induced infiltration of inflammatory cells (neutrophils, eosinophils, and monocytes), the increase in the levels of OVA-specific IgE and Th2 cytokines (IL-4 and IL-5), the production of mucin in the airways, and airway subepithelial fibrosis. Together, the results suggest that the PAI-1 inhibitor TM5275 may have therapeutic potential for asthma through suppressing eosinophilic allergic response and ameliorating airway remodeling. PMID:26702150

  1. Therapeutic potential of an orally effective small molecule inhibitor of plasminogen activator inhibitor for asthma.

    PubMed

    Liu, Rui-Ming; Eldridge, Stephanie; Watanabe, Nobuo; Deshane, Jessy; Kuo, Hui-Chien; Jiang, Chunsun; Wang, Yong; Liu, Gang; Schwiebert, Lisa; Miyata, Toshio; Thannickal, Victor J

    2016-02-15

    Asthma is one of the most common respiratory diseases. Although progress has been made in our understanding of airway pathology and many drugs are available to relieve asthma symptoms, there is no cure for chronic asthma. Plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of tissue-type and urokinase-type plasminogen activators, has pleiotropic functions besides suppression of fibrinolysis. In this study, we show that administration of TM5275, an orally effective small-molecule PAI-1 inhibitor, 25 days after ovalbumin (OVA) sensitization-challenge, significantly ameliorated airway hyperresponsiveness in an OVA-induced chronic asthma model. Furthermore, we show that TM5275 administration significantly attenuated OVA-induced infiltration of inflammatory cells (neutrophils, eosinophils, and monocytes), the increase in the levels of OVA-specific IgE and Th2 cytokines (IL-4 and IL-5), the production of mucin in the airways, and airway subepithelial fibrosis. Together, the results suggest that the PAI-1 inhibitor TM5275 may have therapeutic potential for asthma through suppressing eosinophilic allergic response and ameliorating airway remodeling.

  2. PAI-1 over-expression decreases experimental post-thrombotic vein wall fibrosis by a non-vitronectin dependent mechanism

    PubMed Central

    Obi, Andrea T.; Diaz, Jose A.; Ballard-Lipka, Nicole L.; Roelofs, Karen J.; Farris, Diana M.; Lawrence, Daniel A.; Wakefield, Thomas W.; Henke, Peter K.

    2014-01-01

    SUMMARY Background Factors associated with post-thrombotic syndrome are known clinically, but the underlying cellular processes at the vein wall are not well-delineated. Prior work suggests that vein wall damage does not correlate with thrombus resolution, but rather with plasminogen activator-1 (PAI-1) and matrix metalloproteinase (MMP) activity. Objective We hypothesized that PAI-1 would confer post venous thrombosis (VT) vein wall protection via a Vitronectin (Vn) dependent mechanism. Methods A stasis model of VT was used with harvest over 2 weeks, in wild type (WT), Vn−/−, and PAI-1 overexpressing mice (PAI-1 Tg). Results PAI-1 Tg mice had larger VT at 6 and 14 days, compared to controls, but Vn−/−mice had no alteration of VT resolution. Gene deletion of Vn resulted in increased, rather than expected decrease in circulating PAI-1 activity. While both Vn−/− and PAI-1 Tg had attenuated intimal fibrosis, PAI-1 Tg had significantly less vein wall collagen and a compensatory increase in collagen III gene expression. Both Vn−/− and PAI-1 Tg vein wall had less monocyte chemotactic factor-1, and fewer macrophages (F4/80), with significantly less MMP-2 activity and decreased TIMP-1 antigen. Ex vivo assessment of TGFβ mediated fibrotic response showed that PAI-1 Tg vein walls had increased profibrotic gene expression (collagen I, III, MMP-2 and α-SMA) as compared with controls, opposite of the in vivo response. Conclusions The absence of Vn increases circulating PAI-1, which positively modulates vein wall fibrosis in a dose-dependent manner. Translationally, PAI-1 elevation may decrease vein wall damage after DVT, perhaps by decreasing macrophage-mediated activities. PMID:24943740

  3. The Omptins of Yersinia pestis and Salmonella enterica Cleave the Reactive Center Loop of Plasminogen Activator Inhibitor 1▿

    PubMed Central

    Haiko, Johanna; Laakkonen, Liisa; Juuti, Katri; Kalkkinen, Nisse; Korhonen, Timo K.

    2010-01-01

    Plasminogen activator inhibitor 1 (PAI-1) is a serine protease inhibitor (serpin) and a key molecule that regulates fibrinolysis by inactivating human plasminogen activators. Here we show that two important human pathogens, the plague bacterium Yersinia pestis and the enteropathogen Salmonella enterica serovar Typhimurium, inactivate PAI-1 by cleaving the R346-M347 bait peptide bond in the reactive center loop. No cleavage of PAI-1 was detected with Yersinia pseudotuberculosis, an oral/fecal pathogen from which Y. pestis has evolved, or with Escherichia coli. The cleavage and inactivation of PAI-1 were mediated by the outer membrane proteases plasminogen activator Pla of Y. pestis and PgtE protease of S. enterica, which belong to the omptin family of transmembrane endopeptidases identified in Gram-negative bacteria. Cleavage of PAI-1 was also detected with the omptins Epo of Erwinia pyrifoliae and Kop of Klebsiella pneumoniae, which both belong to the same omptin subfamily as Pla and PgtE, whereas no cleavage of PAI-1 was detected with omptins of Shigella flexneri or E. coli or the Yersinia chromosomal omptins, which belong to other omptin subfamilies. The results reveal a novel serpinolytic mechanism by which enterobacterial species expressing omptins of the Pla subfamily bypass normal control of host proteolysis. PMID:20639337

  4. Cross-talk between human mast cells and bronchial epithelial cells in plasminogen activator inhibitor-1 production via transforming growth factor-β1.

    PubMed

    Cho, Seong H; Lee, Sun H; Kato, Atsushi; Takabayashi, Tetsuji; Kulka, Marianna; Shin, Soon C; Schleimer, Robert P

    2015-01-01

    Previous reports suggest that plasminogen activator inhibitor-1 (PAI-1) promotes airway remodeling and that human and mouse mast cells (MCs) are an important source of PAI-1. In the present study we investigated MC-epithelial cell (EC) interactions in the production of PAI-1. We stimulated the human MC line LAD2 with IgE-receptor cross-linking and collected the supernatants. We incubated the human bronchial EC line BEAS-2B with the LAD2 supernatants and measured the level of PAI-1. When the supernatants from IgE-stimulated LAD2 were added to BEAS-2B, there was a significant enhancement of PAI-1 production by BEAS-2B. When we treated the MC supernatants with a transforming growth factor (TGF)-β1 neutralizing antibody, the MC-derived induction of PAI-1 from BEAS-2B was completely abrogated. Although TGF-β1 mRNA was constitutively expressed in resting LAD2, it was not highly induced by IgE-mediated stimulation. Nonetheless, active TGF-β1 protein was significantly increased in LAD2 after IgE-mediated stimulation. Active TGF-β1 produced by primary cultured human MCs was significantly reduced in the presence of a chymase inhibitor, suggesting a role of MC chymase as an activator of latent TGF-β1. This study indicates that stimulation of human MCs by IgE receptor cross-linking triggers activation of TGF-β1, at least in part via chymase, which in turn induces the production of PAI-1 by bronchial ECs. Our data suggest that human MCs may play an important role in airway remodeling in asthma as a direct source of PAI-1 and by activating bronchial ECs to produce further PAI-1 via a TGF-β1-mediated activation pathway.

  5. Association between plasminogen activator inhibitor-1 4G/5G gene polymorphism and immunoglobulin A nephropathy susceptibility.

    PubMed

    Zhou, Tian-Biao; Jiang, Zong-Pei

    2015-02-01

    The association between plasminogen activator inhibitor-1 (PAI-1) 4 G/5 G gene polymorphism and immunoglobulin A nephropathy (IgAN) risk is still controversial. A meta-analysis was performed to evaluate the association between PAI-1 4 G/5 G gene polymorphism and IgAN susceptibility. A predefined literature search and selection of eligible relevant studies were performed to collect data from electronic database. Four articles were identified for the analysis of association between PAI-1 4 G/5 G gene polymorphism and IgAN risk. 4 G allele was not associated with IgAN susceptibility in overall populations and in Asians. Furthermore, 4 G/4 G and 5 G/5 G genotype were not associated with IgAN for overall populations, Asians. In conclusion, PAI-1 4 G/5 G gene polymorphism was not associated with IgAN risk in overall populations and in Asians. However, more studies should be performed in the future.

  6. Decreased expression of the plasminogen activator inhibitor type 1 is involved in degradation of extracellular matrix surrounding cervical cancer stem cells.

    PubMed

    Sato, Masakazu; Kawana, Kei; Adachi, Katsuyuki; Fujimoto, Asaha; Yoshida, Mitsuyo; Nakamura, Hiroe; Nishida, Haruka; Inoue, Tomoko; Taguchi, Ayumi; Takahashi, Juri; Kojima, Satoko; Yamashita, Aki; Tomio, Kensuke; Nagamatsu, Takeshi; Wada-Hiraike, Osamu; Oda, Katsutoshi; Osuga, Yutaka; Fujii, Tomoyuki

    2016-02-01

    The plasminogen activator (PA) system consists of plasminogen activator inhibitor type 1 (PAI-1), urokinase-type plasminogen activator and its receptor (uPA and uPAR). PAI-1 inhibits the activation of uPA (which converts plasminogen to plasmin), and is involved in cancer invasion and metastasis, by remodeling the extracellular matrix (ECM) through regulating plasmin. Cancer stem cells (CSCs) are a small subset of cells within tumors, and are thought to be involved in tumor recurrence and metastasis. Considering these facts, we investigated the relationship between PAI-1 and cervical CSCs. We used ALDH1 as a marker of cervical CSCs. First, we demonstrated that culturing ALDH1-high cells and ALDH-low cells on collagen IV-coted plates increased their expression of active PAI-1 (ELISA), and these increases were suggested to be at mRNA expression levels (RT-qPCR). Secondly, we demonstrated PAI-1 was indeed involved in the ECM maintenance. With gelatin zymography assays, we found that ALDH1-high cells and ALDH-low cells expressed pro-matrix metalloproteinase-2 (pro-MMP-2) irrespective of their coatings. With gelatinase/collagenase assay kit, we confirmed that collagenase activity was increased when ALDH1-low cells were exposed to TM5275, a small molecule inhibitor of PAI-1. Putting the data together, we hypothesized that cancer cells adhered to basal membrane secrete abundant PAI-1, on the other hand, cancer cells (especially CSCs rather than non-CSCs) distant from basal membrane secrete less PAI-1, which makes the ECM surrounding CSCs more susceptible to degradation. Our study could be an explanation of conflicting reports, where some researchers found negative impacts of PAI-1 expression on clinical outcomes and others not, by considering the concept of CSCs.

  7. Plasminogen activator inhibitor-1 synthesis in the human hepatoma cell line Hep G2. Metformin inhibits the stimulating effect of insulin.

    PubMed Central

    Anfosso, F; Chomiki, N; Alessi, M C; Vague, P; Juhan-Vague, I

    1993-01-01

    High plasma plasminogen activator inhibitor-1 (PAI-1) activity is associated with insulin resistance and is correlated with hyperinsulinemia. The cellular origin of plasma PAI-1 in insulin resistance is not known. The hepatoma cell line Hep G2 has been shown to synthesize PAI-1 in response to insulin. The aim of this study was to analyze the insulin-mediated response of PAI-1 and lipid synthesis in Hep G2 cells after producing an insulin-resistant state by decreasing insulin receptor numbers. The effect of metformin, a dimethyl-substituted biguanide, known to lower plasma insulin and PAI-1 levels in vivo was concomitantly evaluated. Preincubation by an 18-h exposure of Hep G2 cells to 10(-7) M insulin aimed at reducing the number of insulin receptors, was followed by a subsequent 24-h stimulation with 10(-9) M insulin. The decrease in insulin receptors was accompanied as expected, by a reduction in [14C]acetate incorporation, an index of lipid synthesis, whereas PAI-1 secretion and PAI-1 mRNA expression were enhanced. The addition of metformin did not modify the effect of insulin on insulin receptors or [14C]acetate incorporation. In contrast, the drug (10(-4) M) inhibited insulin-mediated PAI-1 synthesis. The results indicate that PAI-1 synthesis in presence of insulin is markedly increased in down-regulated cells, and that metformin inhibits this effect by acting at the cellular level. These in vitro data are relevant with those found in vivo in insulin-resistant patients. Hep G2 cells may be a suitable model to study PAI-1 regulation in response to hyperinsulinemia. Images PMID:8387542

  8. Irradiation-Induced Regulation of Plasminogen Activator Inhibitor Type-1 and Vascular Endothelial Growth Factor in Six Human Squamous Cell Carcinoma Lines of the Head and Neck

    SciTech Connect

    Artman, Tuuli; Schilling, Daniela; Multhoff, Gabriele

    2010-02-01

    Purpose: It has been shown that plasminogen activator inhibitor type-1 (PAI-1) and vascular endothelial growth factor (VEGF) are involved in neo-angiogenesis. The aim of this study was to investigate the irradiation-induced regulation of PAI-1 and VEGF in squamous cell carcinomas of the head and neck (SCCHN) cell lines of varying radiation sensitivity. Methods and Materials: Six cell lines derived from SCCHN were investigated in vitro. The colorimetric AlamarBlue assay was used to detect metabolic activity of cell lines during irradiation as a surrogate marker for radiation sensitivity. PAI-1 and VEGF secretion levels were measured by enzyme-linked immunosorbent assay 24, 48, and 72 h after irradiation with 0, 2, 6, and 10 Gy. The direct radioprotective effect of exogenous PAI-1 was measured using the clonogenic assay. For regulation studies, transforming growth factor-beta1 (TGF-beta1), hypoxia-inducible factor-1alpha (HIF-1alpha), hypoxia-inducible factor-2alpha (HIF-2alpha), or both HIF-1alpha and HIF-2alpha were downregulated using siRNA. Results: Although baseline levels varied greatly, irradiation led to a comparable dose-dependent increase in PAI-1 and VEGF secretion in all six cell lines. Addition of exogenous stable PAI-1 to the low PAI-1-expressing cell lines, XF354 and FaDu, did not lead to a radioprotective effect. Downregulation of TGF-beta1 significantly decreased VEGF secretion in radiation-sensitive XF354 cells, and downregulation of HIF-1alpha and HIF-2alpha reduced PAI-1 and VEGF secretion in radiation-resistant SAS cells. Conclusions: Irradiation dose-dependently increased PAI-1 and VEGF secretion in all SCCHN cell lines tested regardless of their basal levels and radiation sensitivity. In addition, TGF-beta1 and HIF-1alpha could be partly responsible for VEGF and PAI-1 upregulation after irradiation.

  9. Relationship between cathepsin D, urokinase, and plasminogen activator inhibitors in malignant vs benign breast tumours.

    PubMed Central

    Foucré, D.; Bouchet, C.; Hacène, K.; Pourreau-Schneider, N.; Gentile, A.; Martin, P. M.; Desplaces, A.; Oglobine, J.

    1991-01-01

    The concentrations of cathepsin D (Cath D), urokinase (uPA) and two plasminogen activator inhibitors (PAI-1 and PAI-2) were analysed in the cytosols of 130 human mammary tumours (43 benign tumours and 87 primary and unilateral breast carcinomas). uPA, PAI-1 and PAI-2 levels were measured by antigenic immunoassays and Cath D by immunoradiometric assay. The median levels of the four parameters were significantly higher in the malignant tumours than in the benign ones. Cath D and uPA increases were 4-fold and 5-fold respectively. PAI-1 and PAI-2 increases were much more important, 74-fold and 29-fold respectively. In malignant tumours, median levels of Cath D and uPA did not vary according to classical prognostic factors (histologic grade, presence or absence of axillary lymph nodes, steroid receptors, UICC stage, tumour size, age, and menopausal status). However, PAI-1 decreased in ER+ and PR+ tumours and PAI-2 increased in menopausal women's tumours. When Cath D, uPA, PAI-1 and PAI-2 levels in malignant tumours were compared, positive correlations were found for all combinations. The implication of plasminogen activator inhibitors in the phenomenon was surprising and merits further investigation using tools other than global antigen measurements in tumours. PMID:1931618

  10. Effect of Fagonia arabica on thrombin induced release of t-PA and complex of PAI-1 tPA in cultured HUVE cells.

    PubMed

    Aloni, Prutha D; Nayak, Amit R; Chaurasia, Sweta R; Deopujari, Jayant Y; Chourasia, Chhaya; Purohit, Hemant J; Taori, Girdhar M; Daginawala, Hatim F; Kashyap, Rajpal S

    2016-07-01

    Fagonia arabica (FA) possesses a thrombolytic property which has been earlier reported in our laboratory. Current study was undertaken to investigate the effect of aqueous extract of FA on thrombin-induced tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) release from cultured human umbilical vein endothelial cell line (HUVE) for studying its clot lytic activity. For this, establishment of cell line model has been done by isolating the cells from human umbilical cord. Cell toxicity was evaluated using XTT assay. Estimation of t-PA and PAI-1 t-PA complex were done using ELISA technique. Thrombin treatment induces the t-PA and PAI-1 release from HUVE cell line, and FA treatment was found to antagonize the thrombin induced t-PA and PAI-1 release. Our preliminary results suggest that FA may be used as an alternative to thrombolytic drug. However, study demands further experiments using animal model of thrombosis to establish the role of FA as a novel thrombolytic drug. PMID:27419084

  11. Plasminogen activator inhibitor-1 promotes synaptogenesis and protects against aβ(1-42)-induced neurotoxicity in primary cultured hippocampal neurons.

    PubMed

    Cho, Harim; Joo, Yuyoung; Kim, Seonghan; Woo, Ran-Sook; Lee, Sang Hyung; Kim, Hye-Sun

    2013-01-01

    Plasminogen activator inhibitor-1 (PAI-1) is a soluble factor that is released from astrocytes, the most abundant type of glial cell in the brain. PAI-1 was initially identified as inhibiting two types of plasminogen activators, that is, tissue-type plasminogen and urokinase activators that are known to lead to the proteolytic degradation of the extracellular matrix. Recently, PAI-1 was reported to mediate the neuroprotective activity of transforming growth factor-β1 against N-methyl-D-aspartate receptor-mediated excitotoxicity and to be involved in angiogenesis following ischemic stroke, independently of the effects via the inhibition of tissue-type plasminogen and urokinase-type plasminogen activators. In this study, we examined whether PAI-1 influences synaptogenesis and neurotoxicity induced by amyloid beta peptide(1-42) (Aß(1-42)) in rat primary hippocampal neurons. Using immunostaining, treatment with PAI-1 for 24 h was found to significantly upregulate synaptophysin, postsynaptic density-95, and the polysialylated form of neural cell adhesion molecule, compared to treatment with vehicle alone. In addition, PAI-1 has neuroprotective effects against Aβ(1-42)-induced cytotoxicity in rat primary cultured hippocampal neurons. Taken together, our results suggest that PAI-1 has therapeutic potential in Alzheimer's disease by promoting synaptogenesis and by demonstrating neuroprotective effects against Aβ(1-42)-oligomer-induced neurotoxicity in rat primary cultured hippocampal neurons.

  12. Plasminogen activator inhibitor-1 enhances radioresistance and aggressiveness of non-small cell lung cancer cells

    PubMed Central

    Youn, HyeSook; Kim, Joong Sun; Youn, BuHyun

    2016-01-01

    Acquired resistance of tumor cells during treatment limits the clinical efficacy of radiotherapy. Recent studies to investigate acquired resistance under treatment have focused on intercellular communication because it promotes survival and aggressiveness of tumor cells, causing therapy failure and tumor relapse. Accordingly, a better understanding of the functional communication between subpopulations of cells within a tumor is essential to development of effective cancer treatment strategies. Here, we found that conditioned media (CM) from radioresistant non-small cell lung cancer (NSCLC) cells increased survival of radiosensitive cells. Comparative proteomics analysis revealed plasminogen activator inhibitor-1 (PAI-1) as a key molecule in the secretome that acts as an extracellular signaling trigger to strengthen resistance to radiation. Our results revealed that expression and secretion of PAI-1 in radioresistant cells was increased by radiation-induced transcription factors, including p53, HIF-1α, and Smad3. When CM from radioresistant cells was applied to radiosensitive cells, extracellular PAI-1 activated the AKT and ERK1/2 signaling pathway and inhibited caspase-3 activity. Our study also proposed that PAI-1 activates the signaling pathway in radiosensitive cells via extracellular interaction with its binding partners, not clathrin-mediated endocytosis. Furthermore, secreted PAI-1 increased cell migration capacity and expression of EMT markers in vitro and in vivo. Taken together, our findings demonstrate that PAI-1 secreted from radioresistant NSCLC cells reduced radiosensitivity of nearby cells in a paracrine manner, indicating that functional inhibition of PAI-1 signaling has therapeutic potential because it prevents sensitive cells from acquiring radioresistance. PMID:27004408

  13. Role of ACE and PAI-1 Polymorphisms in the Development and Progression of Diabetic Retinopathy.

    PubMed

    Saleem, Saba; Azam, Aisha; Maqsood, Sundus Ijaz; Muslim, Irfan; Bashir, Shaheena; Fazal, Nosheen; Riaz, Moeen; Ali, Syeda Hafiza Benish; Niazi, Muhammad Khizar; Ishaq, Mazhar; Waheed, Nadia Khalida; Qamar, Raheel; Azam, Maleeha

    2015-01-01

    In the present study we determined the association of angiotensin converting enzyme (ACE) and plasminogen activator inhibitor-1 (PAI-1) gene polymorphisms with diabetic retinopathy (DR) and its sub-clinical classes in Pakistani type 2 diabetic patients. A total of 353 diabetic subjects including 160 DR and 193 diabetic non retinopathy (DNR) as well as 198 healthy controls were genotyped by allele specific polymerase chain reaction (PCR) for ACE Insertion/Deletion (ID) polymorphism, rs4646994 in intron 16 and PAI-1 4G/5G (deletion/insertion) polymorphism, rs1799768 in promoter region of the gene. To statistically assess the genotype-phenotype association, multivariate logistic regression analysis was applied to the genotype data of DR, DNR and control individuals as well as the subtypes of DR. The ACE genotype ID was found to be significantly associated with DR (p = 0.009, odds ratio (OR) 1.870 [95% confidence interval (CI) = 1.04-3.36]) and its sub-clinical class non-proliferative DR (NPDR) (p = 0.006, OR 2.250 [95% CI = 1.098-4.620]), while PAI polymorphism did not show any association with DR in the current cohort. In conclusion in Pakistani population the ACE ID polymorphism was observed to be significantly associated with DR and NPDR, but not with the severe form of the disease i.e. proliferative DR (PDR). PMID:26658948

  14. Hyperthermia stimulates plasminogen activator inhibitor type 1 expression in human umbilical vein endothelial cells in vitro.

    PubMed Central

    Wojta, J.; Holzer, M.; Hufnagl, P.; Christ, G.; Hoover, R. L.; Binder, B. R.

    1991-01-01

    The effect of exposure to hyperthermia on the fibrinolytic potential of human umbilical vein endothelial cells (HUVEC) in culture was studied. HUVEC responded to exposure to 42 degrees C with a time-dependent increase in plasminogen activator inhibitor type 1 (PAI-1) activity and antigen accompanied by a four- to fivefold increase in PAI-1 specific m-RNA and a decrease in tissue-type plasminogen activator (t-PA) antigen. The effect of 8 hours exposure to hyperthermia on PAI-1 activity and antigen could not be reversed by reexposure of the cells to 37 degrees C for 24 hours as evidenced by continuously increased amounts of PAI-1 released into the conditioned media. t-PA release, however, decreased during the 24-hour period at 37 degrees C after exposure to hyperthermia. No difference in PAI-1 antigen present in the extracellular matrix of heat treated HUVEC as compared to HUVEC kept at 37 degrees C could be found. Our data supports the idea that hyperthermia is one stress factor that influences the fibrinolytic potential of endothelial cells. Images Figure 6 PMID:1928306

  15. The effects of polymorphisms in genes from the renin-angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels are dependent on environmental context.

    PubMed

    Asselbergs, Folkert W; Williams, Scott M; Hebert, Patricia R; Coffey, Christopher S; Hillege, Hans L; Snieder, Harold; Navis, Gerjan; Vaughan, Douglas E; van Gilst, Wiek H; Moore, Jason H

    2007-11-01

    Thrombosis is a key factor in the pathophysiology of cardiovascular disease. Important biochemical constituents of the fibrinolytic system, affecting thrombosis, include tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1). Both t-PA and PAI-1 are determined by multiple genetic and environmental factors. We aimed to investigate whether the effects of polymorphism in genes from the renin-angiotensin, bradykinin, and fibrinolytic systems on t-PA or PAI-1 levels are dependent on environmental factors in a large population-based sample from the PREVEND study in Groningen, The Netherlands (n = 2,527). We found strong evidence (P PAI-1 in males. Only suggestive evidence (P PAI-1 levels in females. Another consistent finding was evidence for an interaction between bradykinin receptor (BDKRB2) gene polymorphisms and body size as measured by body mass index and/or waist-hip-ratio. For each gender and for both t-PA and PAI-1 there was at least one BDKRB2-body size combination that exhibited suggestive (P PAI-1 is dependent on the environmental context such as body size and alcohol use. The present study emphasizes the importance of including environmental factors in genetic analyses to fully comprehend the genetic architecture of a specific trait.

  16. Immobilization of the distal hinge in the labile serpin plasminogen activator inhibitor 1: identification of a transition state with distinct conformational and functional properties.

    PubMed

    De Taeye, Bart; Compernolle, Griet; Dewilde, Maarten; Biesemans, Wouter; Declerck, Paul J

    2003-06-27

    The serpin plasminogen activator inhibitor-1 (PAI-1) plays an important role in the regulation of the fibrinolytic activity in blood. In plasma, PAI-1 circulates mainly in the active conformation. However, PAI-1 spontaneously converts to a latent conformation. This conversion comprises drastic conformational changes in both the distal and the proximal hinge region of the reactive center loop. To study the functional and conformational rearrangements associated solely with the mobility of the proximal hinge, disulfide bonds were introduced to immobilize the distal hinge region. These mutants exhibited specific activities comparable with that of PAI-1-wt. However, the engineered disulfide bond had a major effect on the conformational and associated functional transitions. Strikingly, in contrast to PAI-1-wt, inactivation of these mutants yielded a virtually complete conversion to a substrate-like conformation. Comparison of the digestion pattern (with trypsin and elastase) of the mutants and PAI-1-wt revealed that the inactivated mutants have a conformation differing from that of latent and active PAI-1-wt. Unique trypsin-susceptible cleavage sites arose upon inactivation of these mutants. The localization of these exposed residues provides evidence that a displacement of alphahF has occurred, indicating that the proximal hinge is partly inserted between s3A and s5A. In conclusion, immobilization of the distal hinge region in PAI-1 allowed the identification of an "intermediate" conformation characterized by a partial insertion of the proximal hinge region. We hypothesize that locking PAI-1 in this transition state between active and latent conformations is associated with a displacement of alphahF, subsequently resulting in substrate behavior.

  17. Cardiovascular-renal complications and the possible role of plasminogen activator inhibitor: a review.

    PubMed

    D'Elia, John A; Bayliss, George; Gleason, Ray E; Weinrauch, Larry A

    2016-10-01

    Since angiotensin increases the expression of plasminogen activator inhibitor (PAI), mechanisms associated with an actively functioning renin-angiotensin-aldosterone system can be expected to be associated with increased PAI-1 expression. These mechanisms are present not only in common conditions resulting in glomerulosclerosis associated with aging, diabetes or genetic mutations, but also in autoimmune disease (like scleroderma and lupus), radiation injury, cyclosporine toxicity, allograft nephropathy and ureteral obstruction. While the renin-angiotensin-aldosterone system and growth factors, such as transforming growth factor-beta (TGF-β), are almost always part of the process, there are rare experimental observations of PAI-1 expression without their interaction. Here we review the literature on PAI-1 and its role in vascular, fibrotic and oxidative injury as well as work suggesting potential areas of intervention in the pathogenesis of multiple disorders. PMID:27679717

  18. Cardiovascular-renal complications and the possible role of plasminogen activator inhibitor: a review

    PubMed Central

    D'Elia, John A.; Bayliss, George; Gleason, Ray E.; Weinrauch, Larry A.

    2016-01-01

    Since angiotensin increases the expression of plasminogen activator inhibitor (PAI), mechanisms associated with an actively functioning renin–angiotensin–aldosterone system can be expected to be associated with increased PAI-1 expression. These mechanisms are present not only in common conditions resulting in glomerulosclerosis associated with aging, diabetes or genetic mutations, but also in autoimmune disease (like scleroderma and lupus), radiation injury, cyclosporine toxicity, allograft nephropathy and ureteral obstruction. While the renin–angiotensin–aldosterone system and growth factors, such as transforming growth factor-beta (TGF-β), are almost always part of the process, there are rare experimental observations of PAI-1 expression without their interaction. Here we review the literature on PAI-1 and its role in vascular, fibrotic and oxidative injury as well as work suggesting potential areas of intervention in the pathogenesis of multiple disorders. PMID:27679717

  19. Cardiovascular-renal complications and the possible role of plasminogen activator inhibitor: a review

    PubMed Central

    D'Elia, John A.; Bayliss, George; Gleason, Ray E.; Weinrauch, Larry A.

    2016-01-01

    Since angiotensin increases the expression of plasminogen activator inhibitor (PAI), mechanisms associated with an actively functioning renin–angiotensin–aldosterone system can be expected to be associated with increased PAI-1 expression. These mechanisms are present not only in common conditions resulting in glomerulosclerosis associated with aging, diabetes or genetic mutations, but also in autoimmune disease (like scleroderma and lupus), radiation injury, cyclosporine toxicity, allograft nephropathy and ureteral obstruction. While the renin–angiotensin–aldosterone system and growth factors, such as transforming growth factor-beta (TGF-β), are almost always part of the process, there are rare experimental observations of PAI-1 expression without their interaction. Here we review the literature on PAI-1 and its role in vascular, fibrotic and oxidative injury as well as work suggesting potential areas of intervention in the pathogenesis of multiple disorders.

  20. The angiotensin receptor blocker, azilsartan medoxomil (TAK-491), suppresses vascular wall expression of plasminogen activator inhibitor type-I protein potentially facilitating the stabilization of atherosclerotic plaques.

    PubMed

    French, Christopher J; Zaman, A K M Tarikuz; Sobel, Burton E

    2011-08-01

    Increased expression of plasminogen activator inhibitor type-I (PAI-1) in vessel walls seems to accelerate atherosclerosis. Angiotensin II can increase the synthesis of PAI-1. Inhibition of this process may facilitate migration of vascular smooth muscle cells (VSMCs) stabilizing atherosclerotic plaques. To determine whether the inhibition of the angiotensin II type 1 receptor can blunt the expression of PAI-1 protein in the aortic wall, we administered azilsartan medoxomil (AZL-M), a prodrug of an angiotensin II type 1 receptor blocker developed by the Takeda Pharmaceutical Company Limited, for 16 weeks to ApoE knockout mice on a high fat diet rendered overexpressors of PAI-1 in VSMCs. Homogenates of the pooled aortas from each group were assayed for PAI-1 by enzyme-linked immunosorbent assay. Cellularity of atherosclerotic lesions was assessed by 4',6-diamidino-2-phenylindole staining in sections of aortic lesions, and collagen content in the lesions was quantified by immunohistochemistry. Aortic wall PAI-1 was decreased by each of the 3 dosage regimens of AZL-M (0.1-10 mg/kg). Cellularity and collagen were increased in lesions from mice given AZL-M, consistent with the development of more stable plaques. Accordingly, the suppression of PAI-1 expression by AZL-M may attenuate the evolution of atherosclerotic plaques vulnerable to rupture.

  1. Synergistic and multidimensional regulation of plasminogen activator inhibitor type 1 expression by transforming growth factor type β and epidermal growth factor

    SciTech Connect

    Song, Xiaoling; Thalacker, F.W.; Nilsen-Hamilton, Marit

    2012-04-06

    The major physiological inhibitor of plasminogen activator, type I plasminogen activator inhibitor (PAI-1), controls blood clotting and tissue remodeling events that involve cell migration. Transforming growth factor type β (TGFβ) and epidermal growth factor (EGF) interact synergistically to increase PAI-1 mRNA and protein levels in human HepG2 and mink Mv1Lu cells. Other growth factors that activate tyrosine kinase receptors can substitute for EGF. EGF and TGFβ regulate PAI-1 by synergistically activating transcription, which is further amplified by a decrease in the rate of mRNA degradation, the latter being regulated only by EGF. The combined effect of transcriptional activation and mRNA stabilization results in a rapid 2-order of magnitude increase in the level of PAI-1. TGFβ also increases the sensitivity of the cells to EGF, thereby recruiting the cooperation of EGF at lower than normally effective concentrations. The contribution of EGF to the regulation of PAI-1 involves the MAPK pathway, and the synergistic interface with the TGFβ pathway is downstream of MEK1/2 and involves phosphorylation of neither ERK1/2 nor Smad2/3. Synergism requires the presence of both Smad and AP-1 recognition sites in the promoter. This work demonstrates the existence of a multidimensional cellular mechanism by which EGF and TGFβ are able to promote large and rapid changes in PAI-1 expression.

  2. Plasminogen activator inhibitor-1 regulates infiltration of macrophages into melanoma via phosphorylation of FAK-Tyr⁹²⁵.

    PubMed

    Thapa, Bikash; Koo, Bon-Hun; Kim, Yeon Hyang; Kwon, Hyung-Joo; Kim, Doo-Sik

    2014-08-01

    Tumor-infiltrating macrophages are potential candidates for cancer immunotherapy. However, the detailed molecular mechanism underlying macrophage infiltration into tumors is poorly understood. Based on our previous finding that plasminogen activator inhibitor-1 (PAI-1) enhances vitronectin-dependent migration of macrophages, we investigated the potential role of PAI-1 in macrophage invasion into melanoma. Experimental evidence obtained from spheroid confrontation assay clearly showed that PAI-1 overexpression significantly enhanced the invasion of RAW 264.7 cells into B16F10 melanoma. We further demonstrated that PAI-1 induces phosphorylation of focal adhesion kinase (FAK) at Tyr(925), which, in turn, mediated the invasion of macrophages into the melanoma. This work further illustrates that low-density lipoprotein receptor related-protein 1 (LRP1) is essential for PAI-1-mediated FAK phosphorylation and macrophage invasion into melanoma. In conclusion, our study demonstrates a novel role of PAI-1 in macrophage invasion into melanoma and provides insights into the underlying molecular mechanism.

  3. Plasminogen activator inhibitor-1 and type 2 diabetes: a systematic review and meta-analysis of observational studies

    PubMed Central

    Yarmolinsky, James; Bordin Barbieri, Natália; Weinmann, Tobias; Ziegelmann, Patricia K.; Duncan, Bruce B.; Inês Schmidt, Maria

    2016-01-01

    An emerging body of evidence has implicated plasminogen activator inhibitor-1 (PAI-1) in the development of type 2 diabetes (T2D), though findings have not always been consistent. We systematically reviewed epidemiological studies examining the association of PAI-1 with T2D. EMBASE, PubMed, Web of Science, and the Cochrane Library were searched to identify studies for inclusion. Fifty-two studies (44 cross-sectional with 47 unique analytical comparisons and 8 prospective) were included. In pooled random-effects analyses of prospective studies, a comparison of the top third vs. bottom third of baseline PAI-1 values generated a RR of T2D of 1.67 (95% CI 1.28–2.18) with moderate heterogeneity (I2 = 38%). Additionally, of 47 cross-sectional comparisons, 34(72%) reported significantly elevated PAI-1 among diabetes cases versus controls, 2(4%) reported significantly elevated PAI-1 among controls, and 11(24%) reported null effects. Results from pooled analyses of prospective studies did not differ substantially by study design, length of follow-up, adjustment for various putative confounding factors, or study quality, and were robust to sensitivity analyses. Findings from this systematic review of the available epidemiological literature support a link between PAI-1 and T2D, independent of established diabetes risk factors. Given the moderate size of the association and heterogeneity across studies, future prospective studies are warranted. PMID:26813008

  4. Altered expression of urokinase-type plasminogen activator and plasminogen activator inhibitor in high-risk soft tissue sarcomas.

    PubMed

    Benassi, M S; Ponticelli, F; Azzoni, E; Gamberi, G; Pazzaglia, L; Chiechi, A; Conti, A; Spessotto, P; Scapolan, M; Pignotti, E; Bacchini, P; Picci, P

    2007-09-01

    In recent years, classification of soft-tissue sarcomas (STS) has improved with cytogenetic analyses, but their clinical behavior is still not easily predictable. The aim of this study was to detect alterations in the urokinase-type plasminogen system, involved in tumor growth and invasion, by comparing mRNA levels of its components with those of paired normal tissues, and relating them with patient clinical course. Real-time PCR was performed on human STS cell lines and tissues from highly malignant STS, including leiomyosarcomas and malignant fibrous histiocytomas, to evaluate the expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1). Immunohistochemistry of gene products was also performed. Median mRNA values of all genes studied were higher in tumors than in paired normal tissues. In agreement with data on STS cell lines, significant up-regulation for uPA and PAI-1 genes compared to reference values was seen. Moreover, different levels of expression were related to histotype and metastatic phenotype. There was accordance between uPA mRNA and protein expression, while immunodetection of PAI-1 product was weak and scattered. Clearly, the controversial role of PAI-1 protein requires further biological analyses, but evident involvement of uPA/PAI-1 gene overexpression in STS malignancy may highlight a molecular defect useful in discriminating STS high-risk patients. PMID:17523079

  5. Dynamic changes in plasma tissue plasminogen activator, plasminogen activator inhibitor-1 and beta-thromboglobulin content in ischemic stroke.

    PubMed

    Zhuang, Ping; Wo, Da; Xu, Zeng-Guang; Wei, Wei; Mao, Hui-ming

    2015-07-01

    The aim of this paper is to investigate the corresponding variations of plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) activities, and beta-thromboglobulin (β-TG) content in patients during different stages of ischemic stroke. Ischemic stroke is a common disease among aging people and its occurrence is associated with abnormalities in the fibrinolytic system and platelet function. However, few reports focus on the dynamic changes in the plasma fibrinolytic system and β-TG content in patients with ischemic stroke. Patients were divided into three groups: acute, convalescent and chronic. Plasma t-PA and PAI-1 activities were determined by chromogenic substrate analysis and plasma β-TG content was detected by radioimmunoassay. Patients in the acute stage of ischemic stroke had significantly increased levels of t-PA activity and β-TG content, but PAI-1 activity was significantly decreased. Negative correlations were found between plasma t-PA and PAI-1 activities and between plasma t-PA activity and β-TG content in patients with acute ischemic stroke. There were significant differences in plasma t-PA and PAI-1 activities in the aged control group, as well as in the acute, convalescent and chronic groups. It can be speculated that the increased activity of t-PA in patients during the acute stage was the result of compensatory function, and that the increase in plasma β-TG level not only implies the presence of ischemic stroke but is likely a cause of ischemic stroke. During the later stages of ischemic stroke, greater attention is required in monitoring levels of PAI-1.

  6. Role of hypoxia-inducible transcription factors 1alpha and 2alpha in the regulation of plasminogen activator inhibitor-1 expression in a human trophoblast cell line.

    PubMed

    Meade, E S; Ma, Y Y; Guller, S

    2007-10-01

    The plasminogen activator inhibitors (PAIs) play critical roles in regulating hemostatic and invasive functions of trophoblasts through suppression of plasmin-dependent fibrinolysis and extracellular matrix degradation. The expression of PAI-1 is increased under hypoxic conditions, although the mechanism remains incompletely understood. In the current study we used HTR-8/SVneo cells, a first trimester extravillous trophoblast cell line, and siRNA technology to examine the role of hypoxia-inducible transcription factors (HIFs)-1alpha and -2alpha in the regulation of PAI-1 expression. Using serum-containing and serum-free media culture media it was initially noted that levels of PAI-1, but not PAI-2 protein, were markedly induced by hypoxic (2-3% oxygen) treatment. Under hypoxic conditions, Western blotting revealed that the presence of siRNAs to HIF-1alpha and HIF-2alpha suppressed expression of their respective proteins, whereas treatment with non-targeting and cyclophilin B siRNAs did not. Importantly, incubation with siRNA to HIF-1alpha or HIF-2alpha alone reduced PAI-1 protein levels to a similar extent, with the combined treatment inducing a more profound effect. The presence of HIF siRNAs reduced levels of PAI-1 mRNA as measured by quantitative real-time PCR, indicating that HIF-1alpha and HIF-2 alpha regulate PAI-1 expression at a transcriptional level. These results indicate that both HIF-1alpha and HIF-2alpha play important and similar roles in hypoxia-mediated stimulation of PAI-1 expression in HTR-8/SVneo cells. Our findings provide insight into the physiological regulation of trophoblast PAI-1 expression in early pregnancy when placental oxygen levels are low, as well as a mechanism for over-expression of placental PAI-1 noted in pregnancies with preeclampsia.

  7. Interrelated reduction of chemerin and plasminogen activator inhibitor-1 serum levels in rheumatoid arthritis after interleukin-6 receptor blockade.

    PubMed

    Makrilakis, Konstantinos; Fragiadaki, Kalliopi; Smith, Jacqueline; Sfikakis, Petros P; Kitas, George D

    2015-03-01

    Inflammatory/metabolic factors and imbalance of haemostasis contribute to cardiovascular disease risk in rheumatoid arthritis (RA). Interleukin-6 (IL-6), a cytokine that plays an important role in immune responses, is implicated in its pathogenesis. In this study, the effects of the IL-6 receptor inhibitor, tocilizumab, on serum adipokines and coagulation/fibrinolysis factors in RA patients were examined. Nineteen consecutive patients (18 women, aged 48 ± 9 years) received six monthly infusions of 8 mg/kg tocilizumab for moderate or severe RA. Disease activity/severity, as well as serum levels of chemerin apelin, plasminogen activator inhibitor-1 (PAI-1), interleukin-6 (IL-6), glucose, insulin and lipids were measured at baseline and at 1, 3 and 6 months thereafter. Chemerin and PAI-1 levels decreased significantly from baseline through 3 to 6 months (from 256 ± 79 to 174 ± 12 and 210 ± 85 ng/ml; from 73 ± 27 to 56 ± 22 and 51 ± 28 pg/ml, respectively). Other adipokines did not change, despite increases in adiposity. In multivariate models, significant independent associations were found between baseline chemerin with age, body mass index, remission of disease, HAQ-Di, CRP and PAI-1. Chemerin decrease at 6 months was significantly associated with PAI-1 and IL-6 changes at 6 months. Baseline PAI-1 associated negatively with remission of disease and total cholesterol, while PAI-1 change at 6 months associated with chemerin changes and smoking status. In conclusion, inhibition of IL-6 signaling in RA favorably alters chemerin and PAI-1 levels in an interrelated manner, despite increasing adiposity. This might represent a dual anti-inflammatory and anti-thrombotic/fibrinolytic mechanism of tocilizumab that may reduce cardiovascular event risk in RA patients.

  8. Effect of dipeptidyl peptidase-4 inhibitor, vildagliptin on plasminogen activator inhibitor-1 in patients with diabetes mellitus.

    PubMed

    Tani, Shigemasa; Takahashi, Atsuhiko; Nagao, Ken; Hirayama, Atsushi

    2015-02-15

    Dipeptidyl peptidase-4 (DPP-4) inhibitors may affect the serum levels of plasminogen activator inhibitor-1 (PAI-1) associated with triglyceride (TG) metabolism, which is a prognostic factor for cardiovascular disease, in diabetic patients. We conducted an 8-week, prospective, randomized study in which we assigned type 2 diabetic patients who were inadequately controlled with antidiabetic therapy to the vildagliptin group (50 mg bid, n = 49) or the control group (n = 49). The primary efficacy parameter was the change in the serum level of PAI-1, and the secondary end point was the change in the serum levels of TG-rich lipoproteins. In the vildagliptin group, significant decrease of the serum PAI-1 level by 16.3% (p <0.0001) and significant decreases of the serum TG, remnant-like particle cholesterol, and apolipoprotein B levels by 12.1% (p = 0.002), 13.9% (p = 0.003), and 9.5% (p <0.0001), respectively, were observed. No such changes were observed in the control group. Multivariate regression analyses identified the absolute change from the baseline (Δ) of the PAI-1, but not that of the fasting blood glucose or hemoglobin A1c, as independent predictors of the ΔTG, Δ remnant-like particle cholesterol, and Δ apolipoprotein B. In conclusion, treatment of type 2 diabetes with vildagliptin might prevent the progression of atherosclerotic cardiovascular disease in diabetic patients by decreasing the serum PAI-1 levels and improving TG metabolism.

  9. Plasminogen Activator Inhibitor-1 Antagonist TM5484 Attenuates Demyelination and Axonal Degeneration in a Mice Model of Multiple Sclerosis.

    PubMed

    Pelisch, Nicolas; Dan, Takashi; Ichimura, Atsuhiko; Sekiguchi, Hiroki; Vaughan, Douglas E; van Ypersele de Strihou, Charles; Miyata, Toshio

    2015-01-01

    Multiple sclerosis (MS) is characterized by inflammatory demyelination and deposition of fibrinogen in the central nervous system (CNS). Elevated levels of a critical inhibitor of the mammalian fibrinolitic system, plasminogen activator inhibitor 1 (PAI-1) have been demonstrated in human and animal models of MS. In experimental studies that resemble neuroinflammatory disease, PAI-1 deficient mice display preserved neurological structure and function compared to wild type mice, suggesting a link between the fibrinolytic pathway and MS. We previously identified a series of PAI-1 inhibitors on the basis of the 3-dimensional structure of PAI-1 and on virtual screening. These compounds have been reported to provide a number of in vitro and in vivo benefits but none was tested in CNS disease models because of their limited capacity to penetrate the blood-brain barrier (BBB). The existing candidates were therefore optimized to obtain CNS-penetrant compounds. We performed an in vitro screening using a model of BBB and were able to identify a novel, low molecular PAI-1 inhibitor, TM5484, with the highest penetration ratio among all other candidates. Next, we tested the effects on inflammation and demyelination in an experimental allergic encephalomyelitis mice model. Results were compared to either fingolimod or 6α-methylprednisolone. Oral administration of TM5484 from the onset of signs, ameliorates paralysis, attenuated demyelination, and axonal degeneration in the spinal cord of mice. Furthermore, it modulated the expression of brain-derived neurotrophic factor, which plays a protective role in neurons against various pathological insults, and choline acetyltransferase, a marker of neuronal density. Taken together, these results demonstrate the potential benefits of a novel PAI-1 inhibitor, TM5484, in the treatment of MS.

  10. Plasminogen Activator Inhibitor-1 Antagonist TM5484 Attenuates Demyelination and Axonal Degeneration in a Mice Model of Multiple Sclerosis.

    PubMed

    Pelisch, Nicolas; Dan, Takashi; Ichimura, Atsuhiko; Sekiguchi, Hiroki; Vaughan, Douglas E; van Ypersele de Strihou, Charles; Miyata, Toshio

    2015-01-01

    Multiple sclerosis (MS) is characterized by inflammatory demyelination and deposition of fibrinogen in the central nervous system (CNS). Elevated levels of a critical inhibitor of the mammalian fibrinolitic system, plasminogen activator inhibitor 1 (PAI-1) have been demonstrated in human and animal models of MS. In experimental studies that resemble neuroinflammatory disease, PAI-1 deficient mice display preserved neurological structure and function compared to wild type mice, suggesting a link between the fibrinolytic pathway and MS. We previously identified a series of PAI-1 inhibitors on the basis of the 3-dimensional structure of PAI-1 and on virtual screening. These compounds have been reported to provide a number of in vitro and in vivo benefits but none was tested in CNS disease models because of their limited capacity to penetrate the blood-brain barrier (BBB). The existing candidates were therefore optimized to obtain CNS-penetrant compounds. We performed an in vitro screening using a model of BBB and were able to identify a novel, low molecular PAI-1 inhibitor, TM5484, with the highest penetration ratio among all other candidates. Next, we tested the effects on inflammation and demyelination in an experimental allergic encephalomyelitis mice model. Results were compared to either fingolimod or 6α-methylprednisolone. Oral administration of TM5484 from the onset of signs, ameliorates paralysis, attenuated demyelination, and axonal degeneration in the spinal cord of mice. Furthermore, it modulated the expression of brain-derived neurotrophic factor, which plays a protective role in neurons against various pathological insults, and choline acetyltransferase, a marker of neuronal density. Taken together, these results demonstrate the potential benefits of a novel PAI-1 inhibitor, TM5484, in the treatment of MS. PMID:25915660

  11. High-Resolution structure of the stable plasminogen activator inhibitor type-1 variant 14-1B in its proteinase-cleaved form: A new tool for detailed interaction studies and modeling

    SciTech Connect

    Jensen, J.; Gettins, P.

    2008-10-22

    Wild-type plasminogen activator inhibitor type-1 (PAI-1) rapidly converts to the inactive latent state under conditions of physiological pH and temperature. For in vivo studies of active PAI-1 in cell culture and in vivo model systems, the 14-1B PAI-1 mutant (N150H-K154T-Q319L-M354I), with its stabilized active conformation, has thus become the PAI-1 of choice. As a consequence of the increased stability, the only two forms likely to be encountered are the active or the cleaved form, the latter either free or complexed with target proteinase. We hereby report the first structure of the stable 14-1B PAI-1 variant in its reactive center cleaved form, to a resolution of 2.0 {angstrom}. The >99% complete structure represents the highest resolved structure of free cleaved PAI-1. This high-resolution structure should be of great use for drug target development and for modeling protein-protein interactions such as those of PAI-1 with vitronectin.

  12. Regulatory role of NADPH oxidase in glycated LDL-induced upregulation of plasminogen activator inhibitor-1 and heat shock factor-1 in mouse embryo fibroblasts and diabetic mice.

    PubMed

    Zhao, Ruozhi; Le, Khuong; Moghadasian, Mohammed H; Shen, Garry X

    2013-08-01

    Cardiovascular disease is the predominant cause of death in diabetic patients. Fibroblasts are one of the major types of cells in the heart or vascular wall. Increased levels of glycated low-density lipoprotein (glyLDL) were detected in diabetic patients. Previous studies in our group demonstrated that oxidized LDL increased the amounts of NADPH oxidase (NOX), plasminogen activator inhibitor-1 (PAI-1), and heat shock factor-1 (HSF1) in fibroblasts. This study examined the expression of NOX, PAI-1, and HSF1 in glyLDL-treated wild-type or HSF1-deficient mouse embryo fibroblasts (MEFs) and in leptin receptor-knockout (db/db) diabetic mice. Treatment with physiologically relevant levels of glyLDL increased superoxide and H2O2 release and the levels of NOX4 and p22phox (an essential component of multiple NOX complexes) in wild-type or HSF1-deficient MEFs. The levels of HSF1 and PAI-1 were increased by glyLDL in wild-type MEFs, but not in HSF1-deficient MEFs. Diphenyleneiodonium (a nonspecific NOX inhibitor) or small interfering RNA for p22phox prevented glyLDL-induced increases in the levels of NOX4, HSF1, or PAI-1 in MEFs. The amounts of NOX4, HSF1, and PAI-1 were elevated in hearts of db/db diabetic mice compared to wild-type mice. The results suggest that glyLDL increased the abundance of NOX4 or p22phox via an HSF1-independent pathway, but that of PAI-1 via an HSF1-dependent manner. NOX4 plays a crucial role in glyLDL-induced expression of HSF1 and PAI-1 in mouse fibroblasts. Increased expression of NOX4, HSF1, and PAI-1 was detected in cardiovascular tissue of diabetic mice.

  13. Regulation of plasminogen activator inhibitor-1 expression in endothelial cells with exposure to metal nanoparticles.

    PubMed

    Yu, Min; Mo, Yiqun; Wan, Rong; Chien, Sufan; Zhang, Xing; Zhang, Qunwei

    2010-05-19

    Recent studies demonstrated that exposure to nanoparticles could enhance the adhesion of endothelial cells and modify the membrane structure of vascular endothelium. The endothelium plays an important role in the regulation of fibrinolysis, and imbalance of the fibrinolysis system potential contributes to the development of thrombosis. Plasminogen activator inhibitor-1 (PAI-1) is the most potent endogenous inhibitor of fibrinolysis and is involved in the pathogenesis of several cardiovascular diseases. The aim of this study was to investigate the alteration of PAI-1 expression in mouse pulmonary microvascular endothelial cells (MPMVEC) exposed to the metal nanoparticles that are known to be reactive, and the potential underlying mechanisms. We compared the alteration of PAI-1 expression in MPMVEC exposed to non-toxic doses of nano-size copper (II) oxide (Nano-CuO) and nano-size titanium dioxide (Nano-TiO(2)). Our results showed that Nano-CuO caused a dose- and time-dependent increase in PAI-1 expression. Moreover, exposure of MPMVEC to Nano-CuO caused reactive oxygen species (ROS) generation that was abolished by pre-treatment of cells with ROS scavengers or inhibitors, DPI, NAC and catalase. Exposure of MPMVEC to Nano-CuO also caused a dose- and time-dependent increase in p38 phosphorylation by Western blot. These effects were significantly attenuated when MPMVEC were pre-treated with DPI, NAC and catalase. To further investigate the role of p38 phosphorylation in Nano-CuO-induced PAI-1 overexpression, the p38 inhibitor, SB203580, was used to pre-treat cells prior to Nano-CuO exposure. We found that Nano-CuO-induced overexpression of PAI-1 was attenuated by p38 inhibitor pre-treatment. However, Nano-TiO(2) did not show the same results. Our results suggest that Nano-CuO caused up-regulation of PAI-1 in endothelial cells is mediated by p38 phosphorylation due to oxidative stress. These findings have important implications for understanding the potential health

  14. High-fat diet enhances and plasminogen activator inhibitor-1 deficiency attenuates bone loss in mice with Lewis Lung carcinoma

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study determined the effects of a high-fat diet and plasminogen activator inhibitor-1 deficiency (PAI-1-/-) on bone structure in mice bearing Lewis lung carcinoma (LLC) in lungs. Reduction in bone volume fraction (BV/TV) by 22% and 21%, trabecular number (Tb.N) by 8% and 4% and bone mineral de...

  15. Hypoxia dysregulates the production of adiponectin and plasminogen activator inhibitor-1 independent of reactive oxygen species in adipocytes

    SciTech Connect

    Chen Baoying; Lam, Karen S.L.; Wang Yu; Wu Donghai; Lam, Michael C.; Shen Jiangang; Wong Laiching; Hoo, Ruby L.C.; Zhang Jialiang; Xu Aimin . E-mail: amxu@hkucc.hku.hk

    2006-03-10

    Low plasma levels of adiponectin (hypoadiponectinemia) and elevated circulating concentrations of plasminogen activator inhibitor (PAI)-1 are causally associated with obesity-related insulin resistance and cardiovascular disease. However, the mechanism that mediates the aberrant production of these two adipokines in obesity remains poorly understood. In this study, we investigated the effects of hypoxia and reactive oxygen species (ROS) on production of adiponectin and PAI-1 in 3T3-L1 adipocytes. Quantitative PCR and immunoassays showed that ambient hypoxia markedly suppressed adiponectin mRNA expression and its protein secretion, and increased PAI-1 production in mature adipocytes. Dimethyloxallyl glycine, a stabilizer of hypoxia-inducible factor 1{alpha} (HIF-1{alpha}), mimicked the hypoxia-mediated modulations of these two adipokines. Hypoxia caused a modest elevation of ROS in adipocytes. However, ablation of intracellular ROS by antioxidants failed to alleviate hypoxia-induced aberrant production of adiponectin and PAI-1. On the other hand, the antioxidants could reverse hydrogen peroxide (H{sub 2}O{sub 2})-induced dysregulation of adiponectin and PAI-1 production. H{sub 2}O{sub 2} treatment decreased the expression levels of peroxisome proliferator-activated receptor gamma (PPAR{gamma}) and CCAAT/enhancer binding protein (C/EBP{alpha}), but had no effect on HIF-1{alpha}, whereas hypoxia stabilized HIF-1{alpha} and decreased expression of C/EBP{alpha}, but not PPAR{gamma}. Taken together, these data suggest that hypoxia and ROS decrease adiponectin production and augment PAI-1 expression in adipocytes via distinct signaling pathways. These effects may contribute to hypoadiponectinemia and elevated PAI-1 levels in obesity, type 2 diabetes, and cardiovascular diseases.

  16. Plasminogen activator inhibitor-1 is a critical downstream target of p53 in the induction of replicative senescence.

    PubMed

    Kortlever, Roderik M; Higgins, Paul J; Bernards, René

    2006-08-01

    p53 limits the proliferation of primary diploid fibroblasts by inducing a state of growth arrest named replicative senescence - a process which protects against oncogenic transformation and requires integrity of the p53 tumour suppressor pathway. However, little is known about the downstream target genes of p53 in this growth-limiting response. Here, we report that suppression of the p53 target gene encoding plasminogen activator inhibitor-1 (PAI-1) by RNA interference (RNAi) leads to escape from replicative senescence both in primary mouse embryo fibroblasts and primary human BJ fibroblasts. PAI-1 knockdown results in sustained activation of the PI(3)K-PKB-GSK3beta pathway and nuclear retention of cyclin D1, consistent with a role for PAI-1 in regulating growth factor signalling. In agreement with this, we find that the PI(3)K-PKB-GSK3beta-cyclin D1 pathway is also causally involved in cellular senescence. Conversely, ectopic expression of PAI-1 in proliferating p53-deficient murine or human fibroblasts induces a phenotype displaying all the hallmarks of replicative senescence. Our data indicate that PAI-1 is not merely a marker of senescence, but is both necessary and sufficient for the induction of replicative senescence downstream of p53.

  17. Modulation of plasminogen activator inhibitor-1 in vivo: a new mechanism for the anti-fibrotic effect of renin-angiotensin inhibition.

    PubMed

    Oikawa, T; Freeman, M; Lo, W; Vaughan, D E; Fogo, A

    1997-01-01

    We examined the potential of in vivo linkage of plasminogen activator inhibitor-1 (PAI-1) and angiotensin II (Ang II) in the setting of endothelial injury and sclerosis following radiation injury in the rat. PAI-1 is a major physiological inhibitor of the plasminogen activator (PA)/plasmin system, a key regulator of fibrinolysis and extracellular matrix (ECM) turnover. PAI-1 mRNA expression in the kidney was markedly increased (9-fold) at 12 weeks after irradiation (P < 1.001 vs. normal control). In situ hybridization revealed significant association of PAI-1 expression with sites of glomerular injury (signal intensity in injured vs. intact glomeruli, P < 0.001). Angiotensin converting enzyme inhibitors (ACEI, captopril or enalapril) or angiotensin II receptor antagonist (AIIRA, L158,809) markedly reduced glomerular lesions (thrombosis, mesangiolysis, and sclerosis; sclerosis index, 0 to 4+ scale, 0.49 +/- 0.20 in untreated vs. 0.05 +/- 0.02, 0.02 +/- 0.01, 0.04 +/- 0.02 in captopril, enalapril and AIIRA, respectively, all P < 0.01 vs untreated). Further, ACEI and AIIRA markedly attenuated increased PAI-1 mRNA expression in the irradiated kidney (36, 19 and 20% expression, respectively, for captopril, enalapril and AIIRA, compared to untreated irradiated kidney, P < 0.05, < 0.01, < 0.01). This effect was selective in that neither tissue-type nor urokinase-type PA mRNA expression was affected by these interventions. Thus, we speculate that inhibition of the renin-angiotensin system may ameliorate injury following radiation by accelerating fibrinolysis and ECM degradation, at least in part, via suppression of PAI-1 expression. In summary, inhibition of Ang II, in addition to its known effects on vascular sclerosis, may also by its novel effect to inhibit PAI-1, lessen fibrosis following endothelial/thrombotic injury.

  18. The Dual Diverse Dynamic Reversible Effects of Ankaferd Blood Stopper on EPCR and PAI-1 Inside Vascular Endothelial Cells With and Without LPS Challenge

    PubMed Central

    Karabıyık, Afife; Yılmaz, Erkan; Güleç, Şükrü; Haznedaroğlu, İbrahim; Akar, Nejat

    2012-01-01

    Objective: Ankaferd blood stopper (ABS) is comprised of a mixture of the plants Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia officinarum, and Urtica dioica. ABS is used as a topical hemostatic agent due to its antihemorrhagic effect, yet its hemostatic mechanism of action remains to be investigated. ABS does not affect the levels of coagulation factors II, V, VII, VIII, IX, X, XI and XII. The aim of this study was to investigate the effects of ABS on endothelium and immune response. As such, we evaluated changes in endothelial cell protein C receptor (EPCR) and plasminogen activator inhibitor type-1 (PAI-1) expression inside human umbilical vein endothelial cells (HUVECs) in the presence and absence of lipopolysaccharides (LPSs). Material and Methods: We exposed HUVECs to 10 μL and 100 μL of ABS for 5 min, 25 min, 50 min, 6 h, and 24 h. Additionally, 10 μg mL–1 of LPS was administered for 1 h to observe the effects of LPS challenge on HUVECs, and then the cells were treated with ABS for 5 min, 25 min, 50 min, and 6 h to observe the effects of ABS on HUVECs. Total RNA was isolated from HUVECs and then the level of expression of EPCR and PAI-1 mRNA was measured. Results: Cells were microscopically observed to arise from the surface and adhere to each other following the administration of ABS to HUVECs. Additionally, after 24 h the cells had normal growth and physiology, which suggests that the adhesive cellular effects of ABS might be reversible. ABS had a negative effect on EPCR and PAI-1 expression; the effect in response to 100 µL was greater than that to 10 µL. EPCR and PAI-1 expression increased over time in response to LPS and 10 µL of ABS. EPCR and PAI-1 expression was very low during the first hour of exposure to LPS and 100 µL of ABS, but after 6 h increased to levels similar to those observed in response to LPS and 10 µL of ABS. Conclusion: It was observed that ABS had dual diverse dynamic reversible effects on EPCR and PAI-1

  19. Involvement of RAGE, NADPH oxidase, and Ras/Raf-1 pathway in glycated LDL-induced expression of heat shock factor-1 and plasminogen activator inhibitor-1 in vascular endothelial cells.

    PubMed

    Sangle, Ganesh V; Zhao, Ruozhi; Mizuno, Tooru M; Shen, Garry X

    2010-09-01

    Atherothrombotic cardiovascular diseases are the predominant causes of mortality of diabetic patients. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor for fibrinolysis, and it is also implicated in inflammation and tissue remodeling. Increased levels of PAI-1 and glycated low-density lipoprotein (glyLDL) were detected in patients with diabetes. Previous studies in our laboratory demonstrated that heat shock factor-1 (HSF1) is involved in glyLDL-induced PAI-1 overproduction in vascular endothelial cells (EC). The present study investigated transmembrane signaling mechanisms involved in glyLDL-induced HSF1 and PAI-1 up-regulation in cultured human vascular EC and streptozotocin-induced diabetic mice. Receptor for advanced glycation end products (RAGE) antibody prevented glyLDL-induced increase in the abundance of PAI-1 in EC. GlyLDL significantly increased the translocation of V-Ha-Ras Harvey rat sarcoma viral oncogene homologue (H-Ras) from cytoplasm to membrane compared with LDL. Farnesyltransferase inhibitor-277 or small interference RNA against H-Ras inhibited glyLDL-induced increases in HSF1 and PAI-1 in EC. Treatment with diphenyleneiodonium, a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor, blocked glyLDL-induced translocation of H-Ras, elevated abundances of HSF1 and PAI-1 in EC, and increased release of hydrogen peroxide from EC. Small interference RNA for p22(phox) prevented glyLDL-induced expression of NOX2, HSF1, and PAI-1 in EC. GlyLDL significantly increased V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) phosphorylation. Treatment with Raf-1 inhibitor blocked glyLDL-induced increase of PAI-1 mRNA in EC. The levels of RAGE, H-Ras, NOX4, HSF1, and PAI-1 were increased in hearts of streptozotocin-diabetic mice and positively correlated with plasma glucose. The results suggest that RAGE, NOX, and H-Ras/Raf-1 are implicated in the up-regulation of HSF1 or PAI-1 in vascular EC under diabetes

  20. The vitronectin binding area of plasminogen activator inhibitor-1, mapped by mutagenesis and protection against an inactivating organochemical ligand.

    PubMed

    Jensen, Jan K; Wind, Troels; Andreasen, Peter A

    2002-06-19

    A distinguishing feature of serpins is their ability to undergo a conformational change consisting in insertion of the reactive centre loop (RCL) into beta-sheet A. In the serpin plasminogen activator inhibitor-1 (PAI-1), RCL movements are regulated by vitronectin, having a previously poorly defined binding site lateral to PAI-1's beta-sheet A. Using a novel strategy, based on identification of amino acid residues necessary for vitronectin protection of PAI-1 against inactivation by 4,4'-dianilino-1,1'-bisnaphthyl-5,5'-disulfonic acid, we have defined a vitronectin binding surface spanning 10 residues between alpha-helix F, beta-strand 2A, and alpha-helix E. Our results contribute to elucidating the unique serpin conformational change.

  1. SERPINE1, PAI-1 protein coding gene, methylation levels and epigenetic relationships with adiposity changes in obese subjects with metabolic syndrome features under dietary restriction

    PubMed Central

    Lopez-Legarrea, Patricia; Mansego, Maria Luisa; Zulet, Marian Angeles; Martinez, Jose Alfredo

    2013-01-01

    Plasminogen activator inhibitor 1 (PAI-1) has been associated with metabolic disorders, through different mechanisms, which could involve changes in DNA methylation. This work aimed to assess the potential relationships of the cytosine methylation levels within SERPINE1 gene transcriptional regulatory region, which codes for PAI-1, in peripheral white blood cells with anthropometrical, metabolic and inflammatory features. Forty-six obese subjects with metabolic syndrome features followed Control or Metabolic Syndrome Reduction in Navarra (RESMENA) energy-restricted (−30%E) diets for 8 weeks. SERPINE1 transcriptional regulatory region methylation at baseline was analyzed by a microarray technical. Both dietary strategies reduced anthropometric and biochemical parameters. The Control group significantly reduced plasma PAI-1 concentrations but not the RESMENA group. Participants from both nutritional interventions with higher SERPINE1 methylation levels at baseline showed significantly major reductions in body weight, total fat mass, android fat mass, total cholesterol and triglycerides, as compared with those with lower initial SERPINE1 methylation levels. In conclusion, the DNA methylation levels of SERPINE1 transcriptional regulatory region were associated with some metabolic and anthropometric changes in obese subjects with metabolic syndrome under energy restriction, suggesting a complex epigenetic network in the regulation of this recognized pro-inflammatory marker. (www.clinicaltrials.gov; NCT01087086) PMID:24249967

  2. Association of a PAI-1 Gene Polymorphism and Early Life Infections with Asthma Risk, Exacerbations, and Reduced Lung Function

    PubMed Central

    Kim, Dong Young; Oh, Sam S.; Torgerson, Dara R.; Pino-Yanes, Maria; Hu, Donglei; Sen, Saunak; Huntsman, Scott; Eng, Celeste; Farber, Harold J.; Rodriguez-Cintron, William; Rodriguez-Santana, Jose R.; Serebrisky, Denise; Thyne, Shannon M.; Borrell, Luisa N.; Williams, L. Keoki; DuPont, William; Seibold, Max A.; Burchard, Esteban G.; Avila, Pedro C.; Kumar, Rajesh

    2016-01-01

    Background Plasminogen activator inhibitor-1 (PAI-1) is induced in airways by virus and may mediate asthmatic airway remodeling. We sought to evaluate if genetic variants and early life lower respiratory infections jointly affect asthma risk. Methods We included Latino children, adolescents, and young adults aged 8–21 years (1736 subjects with physician-diagnosed asthma and 1747 healthy controls) from five U.S. centers and Puerto Rico after excluding subjects with incomplete clinical or genetic data. We evaluated the independent and joint effects of a PAI-1 gain of function polymorphism and bronchiolitis / Respiratory Syncytial Virus (RSV) or other lower respiratory infections (LRI) within the first 2 years of life on asthma risk, asthma exacerbations and lung function. Results RSV infection (OR 9.9, 95%CI 4.9–20.2) and other LRI (OR 9.1, 95%CI 7.2–11.5) were independently associated with asthma, but PAI-1 genotype was not. There were joint effects on asthma risk for both genotype-RSV (OR 17.7, 95% CI 6.3–50.2) and genotype-LRI (OR 11.7, 95% CI 8.8–16.4). A joint effect of genotype-RSV resulted in a 3.1-fold increased risk for recurrent asthma hospitalizations. In genotype-respiratory infection joint effect analysis, FEV1% predicted and FEV1/FVC % predicted were further reduced in the genotype-LRI group (β -2.1, 95% CI -4.0 to -0.2; β -2.0, 95% CI -3.1 to -0.8 respectively). Similarly, lower FEV1% predicted was noted in genotype-RSV group (β -3.1, 95% CI -6.1 to -0.2) with a trend for lower FEV1/FVC % predicted. Conclusions A genetic variant of PAI-1 together with early life LRI such as RSV bronchiolitis is associated with an increased risk of asthma, morbidity, and reduced lung function in this Latino population. PMID:27556405

  3. Polymorphisms of PAI-1 and platelet GP Ia may associate with impairment of renal function and thrombocytopenia in Puumala hantavirus infection

    PubMed Central

    Laine, Outi; Joutsi-Korhonen, Lotta; Mäkelä, Satu; Mikkelsson, Jussi; Pessi, Tanja; Tuomisto, Sari; Huhtala, Heini; Libraty, Daniel; Vaheri, Antti; Karhunen, Pekka; Mustonen, Jukka

    2013-01-01

    Introduction Puumala virus (PUUV) infection is a viral hemorrhagic fever with renal syndrome (HFRS) characterized by thrombocytopenia and acute impairment of renal function. We aimed to assess whether genetic polymorphisms of platelet antigens together with those of von Willebrand factor (VWF) and plasminogen activator inhibitor (PAI-1) correlate with disease severity. Patients and methods 172 consecutive hospital-treated patients with serologically confirmed acute PUUV infection were included. Platelet glycoprotein (GP) IIIa T>C (rs5918), GP Ia T>C (rs1126643), GP Ib C>T (rs6065), GP VI T>C (rs1613662), VWF A>G (rs1063856) and PAI-1 A>G (rs2227631) were genotyped. The associations of the rarer alleles with variables reflecting the severity of the disease were analyzed. Results PAI-1 G-carriers had higher maximum creatinine level compared with the non-carriers (median 213 μmol/l, range 60–1499 μmol/l vs. median 122 μmol/l, range 51–1156 μmol/l, p=0.01). The GG-genotypes had higher creatinine levels than GA- and AA-genotypes (medians 249 μmol/l, 204 μmol/l and 122 μmol/l, respectively, p=0.03). Polymorphisms of GP VI and VWF associated with lower creatinine levels during PUUV infection. The minor C-allele of GP Ia associated with lower platelet counts (median 44×109/l, range 20–90×109/l vs median 64×109/l, range 3–238×109/l; p=0.02). Conclusions Polymorphism of PAI-1, a major regulator of fibrinolysis, has an adverse impact on the outcome of kidney function in PUUV-HFRS. Platelet collagen receptor GP Ia polymorphism associates with lower platelet count. PMID:22133274

  4. MnTE-2-PyP reduces prostate cancer growth and metastasis by suppressing p300 activity and p300/HIF-1/CREB binding to the promoter region of the PAI-1 gene.

    PubMed

    Tong, Qiang; Weaver, Michael R; Kosmacek, Elizabeth A; O'Connor, Brian P; Harmacek, Laura; Venkataraman, Sujatha; Oberley-Deegan, Rebecca E

    2016-05-01

    To improve radiation therapy-induced quality of life impairments for prostate cancer patients, the development of radio-protectors is needed. Our previous work has demonstrated that MnTE-2-PyP significantly protects urogenital tissues from radiation-induced damage. So, in order for MnTE-2-PyP to be used clinically as a radio-protector, it is fully necessary to explore the effect of MnTE-2-PyP on human prostate cancer progression. MnTE-2-PyP inhibited prostate cancer growth in the presence and absence of radiation and also inhibited prostate cancer migration and invasion. MnTE-2-PyP altered p300 DNA binding, which resulted in the inhibition of HIF-1β and CREB signaling pathways. Accordingly, we also found that MnTE-2-PyP reduced the expression of three genes regulated by HIF-1β and/or CREB: TGF-β2, FGF-1 and PAI-1. Specifically, MnTE-2-PyP decreased p300 complex binding to a specific HRE motif within the PAI-1 gene promoter region, suppressed H3K9 acetylation, and consequently, repressed PAI-1 expression. Mechanistically, less p300 transcriptional complex binding is not due to the reduction of binding between p300 and HIF-1/CREB transcription factors, but through inhibiting the binding of HIF-1/CREB transcription factors to DNA. Our data provide an in depth mechanism by which MnTE-2-PyP reduces prostate cancer growth and metastasis, which validates the clinical use of MnTE-2-PyP as a radio-protector to enhance treatment outcomes in prostate cancer radiotherapy. PMID:26944191

  5. Modulation of plasminogen activator inhibitor 1 by Triton X-100--identification of two consecutive conformational transitions.

    PubMed

    Gils, A; Declerck, P J

    1998-08-01

    Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily because of its conformational and functional flexibility. In the present study, we have evaluated the influence of the non-ionic detergent Triton X-100 (TX-100) on the functional stability and conformational transitions of PAI-1. At 37 degrees C, TX-100 induced a concentration-dependent decrease of the functional half-life of PAI-1 resulting in half-lives of 177 +/- 54 min (mean +/- SD, n = 3), 19 +/- 2 min, 1.7 +/- 0.3 min and 0.53 +/- 0.03 min in the presence of 0.005, 0.010, 0.020 and 0.2% TX-100, respectively, compared to a half-life of 270 +/- 146 min in the absence of TX-100. Conformational analysis at various time points and at different temperatures (0 degrees C, 25 degrees C, 37 degrees C) revealed that this inactivation proceeds through the formation of a substrate-like intermediate followed by the formation of the latent form. Kinetic evaluation demonstrated that this conversion fits to two consecutive first-order transitions, i.e. active k1--> substrate k2--> latent. The k1 value was strongly dependent on the concentration of TX-100 (e.g. 0.002 +/- 0.0006 s(-1) and 0.029 +/- 0.003 s(-1) for 0.01% and 0.2% TX-100 at 37 degrees C) whereas the conversion of substrate to latent (k2) was virtually independent of the TX-100 concentration (i.e. 0.012 +/- 0.002 s(-1) and 0.011 +/- 0.001 s(-1) for 0.01 and 0.2% TX-100 at 37 degrees C). Experiments with a variety of other non-ionic amphiphilic compounds revealed that the amphiphilic character of the compound is, at least in part, responsible for the observed effects and strongly indicate that the currently reported mechanism of inactivation is of general importance for the conformational transitions in PAI-1. In conclusion, TX- 100 changes the initial conformation of PAI-1 resulting in altered functional properties. This observation allows us to develop a new model for the mechanism involved in the conformational flexibility of

  6. Low-molecular-weight heparin modulates vein wall fibrotic response in a plasminogen activator inhibitor 1-dependent manner

    PubMed Central

    Obi, Andrea T.; Diaz, Jose A.; Ballard-Lipka, Nicole L.; Roelofs, Karen J.; Farris, Diana M.; Lawrence, Daniel A.; Henke, Peter K.; Wakefield, Thomas W.

    2014-01-01

    Background Treatment with low-molecular-weight heparin (LMWH) favorably alters the vein wall response to deep venous thrombosis (DVT), although the mechanisms remain unclear. Previous studies have suggested that LMWH alters the levels of circulating plasminogen activator inhibitor 1 (PAI-1), a known mediator of fibrosis, and may improve endogenous fibrinolysis. We hypothesized that LMWH favorably alters the vein wall response by binding of PAI-1 and acceleration of fibrinolysis. Methods Wild-type and PAI-1 −/− mice underwent treatment with LMWH after induction of occlusive DVT. Vein wall and plasma were harvested and analyzed by enzyme-linked immunosorbent assay, zymography, real-time polymerase chain reaction, and immunohistochemistry. Results Wild-type mice treated with LMWH exhibited diminished vein wall fibrosis (0.6 ± 0.6 vs 1.4 ± 0.2; P < .01; n = 5) and elevation of circulating PAI-1 (1776 ± 342 vs 567 ± 104 ρg/mL; P < .01; n = 5) compared with untreated controls after occlusive DVT. PAI-1−/− mice treated with LMWH were not similarly protected from fibrosis, despite improved thrombus resolution. Treatment with LMWH was associated with decreased intrathrombus interleukin-lβ (68.6 ± 31.0 vs 223.4 ± 28.9 ρg/mg total protein; P < .01; n = 5) but did not alter inflammatory cell recruitment to the vein wall. PAI-1 −/− mice exhibited significantly elevated intrathrombus (257.2 ± 51.5 vs 4.3 ± 3.8 ρg/mg total protein; n = 5) and vein wall interleukin-13 (187.2 ± 57.6 vs 9.9 ± 1.1 ρg/mg total protein; P < .05; n = 5) as well as vein wall F4/80 positively staining monocytes (53 ± 11 vs 16 ± 2 cells/5 high-power fields; P < .05; n = 4). Conclusions LMWH did not accelerate venous thrombosis resolution but did protect against vein wall fibrosis in a PAI-1-dependent manner in an occlusive DVT model. Lack of PAI-1 correlated with accelerated venous thrombosis resolution but no protection from fibrosis. PAI-1 inhibition as a treatment strategy

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed

    Belalcazar, L Maria; Anderson, Andrea M; Lang, Wei; Schwenke, Dawn C; Haffner, Steven M; Yatsuya, Hiroshi; Rushing, Julia; Vitolins, Mara Z; Reeves, Rebecca; Pi-Sunyer, F Xavier; Tracy, Russell P; Ballantyne, Christie M

    2014-11-01

    Plasminogen activator inhibitor 1 (PAI-1) is elevated in obese individuals with type 2 diabetes and may contribute, independently of traditional factors, to increased cardiovascular disease risk. Fiber intake may decrease PAI-1 levels. We examined the associations of fiber intake and its changes with PAI-1 before and during an intensive lifestyle intervention (ILI) for weight loss in 1,701 Look AHEAD (Action for Health in Diabetes) participants with dietary, fitness, and PAI-1 data at baseline and 1 year. Look AHEAD was a randomized cardiovascular disease trial in 5,145 overweight/obese patients with type 2 diabetes, comparing ILI (goal of ≥7% reduction in baseline weight) with a control arm of diabetes support and education. ILI participants were encouraged to consume vegetables, fruits, and grain products low in sugar and fat. At baseline, median fiber intake was 17.9 g/day. Each 8.3 g/day higher fiber intake was associated with a 9.2% lower PAI-1 level (P=0.008); this association persisted after weight and fitness adjustments (P=0.03). Higher baseline intake of fruit (P=0.019) and high-fiber grain and cereal (P=0.029) were related to lower PAI-1 levels. Although successful in improving weight and physical fitness at 1 year, the ILI in Look AHEAD resulted in small increases in fiber intake (4.1 g/day, compared with -2.35 g/day with diabetes support and education) that were not related to PAI-1 change (P=0.34). Only 31.3% of ILI participants (39.8% of women, 19.1% of men) met daily fiber intake recommendations. Increasing fiber intake in overweight/obese individuals with diabetes interested in weight loss is challenging. Future studies evaluating changes in fiber consumption during weight loss interventions are warranted.

  9. PGE2 reduces MMP-14 and increases plasminogen activator inhibitor-1 in cardiac fibroblasts.

    PubMed

    Kassem, Kamal M; Clevenger, Margarette H; Szandzik, David L; Peterson, Edward; Harding, Pamela

    2014-10-01

    Prostaglandin E2 (PGE2) is elevated during cardiac injury and we have previously shown that mice lacking the PGE2 EP4 receptor display dilated cardiomyopathy (DCM) with increased expression of the membrane type matrix metalloproteinase, MMP-14. We thus hypothesized that PGE2 regulates expression of MMP-14 and also affects fibroblast migration. Primary cultures of neonatal rat ventricular fibroblasts (NVFs) were used to test the effects of PGE2. Gene and protein expression was assessed by real time RT-PCR and Western blot, MMP activity was determined by zymography and migration of NVF was assessed by motility in a transwell system. PGE2 reduced expression of MMP-14 and these effects were antagonized by an EP4 antagonist. An EP4 agonist mimicked the effect of PGE2. PGE2 also increased mRNA and protein levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of MMP activation. However, PGE2-stimulation of PAI-1 was mediated by the EP1/EP3 receptor and not EP4. Migration of NVF was assessed by motility in a transwell system. Treatment of NVFs with PGE2 reduced the number of cells migrating toward 10% FCS. Treatment with the EP2 agonist also reduced migration but did not affect MMP-14 expression or PAI-1. Our results suggest that PGE2 utilizes different receptors and mechanisms to ultimately decrease MMP expression and NVF migration.

  10. Involvement of NADPH oxidase in up-regulation of plasminogen activator inhibitor-1 and heat shock factor-1 in mouse embryo fibroblasts induced by oxidized LDL and in apolipoprotein E-deficient mice.

    PubMed

    Zhao, Ruozhi; Moghadasian, Mohammed H; Shen, Garry X

    2011-09-01

    The present study demonstrated that oxidized LDL (oLDL) increased the generation of superoxide and hydrogen peroxide (H(2)O(2)), the abundances of NADPH oxidase (NOX)4, NOX2, p22-phox and lectin-like oLDL receptor-1 (LOX-1) in wild-type or heat shock factor-1 (HSF1)-deficient mouse embryo fibroblasts (MEF). LOX-1 antibody inhibited LDL or oLDL-induced expression of NOX components in MEF. Abundance of HSF1 or plasminogen activator inhibitor-1 (PAI-1) was increased by oLDL in wild-type, but not in HSF1-deficient MEF. Diphenyleneiodonium or siRNA for NOX or p22-phox inhibited oLDL-induced increases of HSF1, PAI-1 and H(2)O(2) in MEF. Increased NOX4, NOX2, LOX1, HSF1 and PAI-1 were detected in aortae and hearts of apolipoprotein E-knockout (apoE-KO) mice compared to controls, which were associated with increased serum cholesterol or plasma PAI-1. The results suggest that NOX is required for oLDL-induced HSF1 or PAI-1 expression in MEF, which was supported by the up-regulation of NOX, LOX-1, HSF1 and PAI-1 in apoE-KO mice.

  11. The Role of Urokinase Plasminogen Activator and Plasmin Activator Inhibitor-1 on Vein Wall Remodeling in Experimental Deep Vein Thrombosis

    PubMed Central

    Baldwin, Joe F.; Sood, Vikram; Elfline, Megan A.; Luke, Cathy E.; Dewyer, Nicholas A.; Diaz, Jose A.; Myers, Dan D.; Wakefield, Thomas; Henke, Peter K.

    2012-01-01

    OBJECTIVE Deep vein thrombosis (DVT) resolution instigates an inflammatory response, resulting in vessel wall damage and scarring. Urokinase-plasminogen activator (uPA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), are integral components of the fibrinolytic system, essential for VT resolution. This study determined the vein wall response when exposed to increased and decreased plasmin activity. Methods A mouse inferior vena cava (IVC) ligation model in uPA −/− or PAI-1 −/− and their genetic wild types (B6/SvEv and C57/BL6, respectively) was used to create stasis thrombi, with tissue harvest at either 8 or 21d. Tissue analysis included gene expression of vascular smooth muscle cells (alpha SMA [αSMA], SM22) and endothelial marker (CD31), by real time PCR, ELISA, matrix metalloproteinase (MMP) -2 and 9 activity by zymography and vein wall collagen by picrosirius red histological analysis. A P < .05 was considered significant. RESULTS Thrombi were significantly larger in both 8d and 21d uPA −/− as compared to WT, and were significantly smaller in both 8 and 21d PAI-1 −/− as compared to WT. Correspondingly, 8d plasmin levels were reduced in half in uPA −/− and increased 3 fold in PAI-1 −/− when compared to respective WT thrombi (P < .05, N = 5 – 6). The endothelial marker CD31 was elevated 2 fold in PAI-1 −/− mice at 8d, but reduced 2.5 fold at 21d in uPA −/− as compared with WT (P = .02, N = 5 – 6), suggesting less endothelial preservation. Vein wall VSMC gene expression showed that 8d and 21d PAI-1 −/− mice had 2.3 and 3.8 fold more SM22 and 1.8 and 2.3 fold more αSMA expression than respective WT (P < .05, N = 5 – 7), as well as 1.8 fold increased αSMA (+) cells (N = 3 – 5, P ≤ .05). No significant difference in MMP2 or 9 activity was found in the PAI-1 −/− mice compared with WT, while 5.4 fold more MMP9 was present in 21d WT than 21d uPA −/− (P = .03, N = 5). Lastly, collagen was ~2 fold

  12. Plasminogen activator inhibitor-1 fused with erythropoietin (EPO) mimetic peptide (EMP) enhances the EPO activity of EMP.

    PubMed

    Kuai, L; Wu, C; Qiu, Q; Zhang, J; Zhou, A; Wang, S; Zhang, H; Song, Q; Liao, S; Han, Y; Liu, J; Ma, Z

    2000-08-01

    Erythropoietin (EPO) mimetic peptide (EMP) encoding sequence was inserted into the gene of plasminogen activator inhibitor-1 (PAI-1) between Ala348 and Pro349 (P2'-P3'), generating a novel gene, PAI-1/EMP (PMP). This was cloned into pET32a expression vector, fused with TrxA peptide in the vector, and a 63-kDa protein was expressed in inclusion bodies with an expression level >50%. The TrxA/PMP protein was purified by Ni-NTA-agarose metal-ligand affinity chromatography to a purity >90%, showing a single, silver-stained band on SDS-PAGE. Using a reticulocyte counting assay, the EPO activity of PMP was determined to be 5,000 IU/mg, 2,500-fold that of EMP.

  13. mRNAs encoding urokinase-type plasminogen activator and plasminogen activator inhibitor-1 are elevated in the mouse brain following kainate-mediated excitation.

    PubMed

    Masos, T; Miskin, R

    1997-07-01

    Urokinase-type plasminogen activator (uPA) is an inducible extracellular serine protease implicated in fibrinolysis and in tissue remodeling. Recently, we have localized uPA mRNA strictly in limbic structures and the parietal cortex of the adult mouse brain. Here, we tested whether the systemic treatment of mice with kainic acid (KA), an amino acid inducing limbic seizures, could elevate in the brain mRNAs encoding uPA and its specific inhibitor, plasminogen activator inhibitor-1 (PAI-1), a major antifibrinolytic agent. Brain sections encompassing the hippocampus were tested through in situ hybridization using radiolabeled riboprobes specific for the two mRNA species. The results showed that KA greatly enhanced both mRNA species in sites of limbic structures and cortex. However, in the hypothalamus and brain blood vessels only PAI-1 mRNA was elevated. Those were also the only two locations where PAI-1 mRNA was detected in the non-treated control brain, although at a low level. For both mRNAs, KA enhancement was first evident 2-4 h after treatment, and it was most prolonged in the hippocampal area, where prominent hybridization signals persisted for three days. Here, both mRNAs were initially elevated in the hilar region of the dentate gyrus and in the molecular and oriens layers; however, PAI-1 mRNA became evident throughout the area, while uPA mRNA became especially pronounced in the CA3/CA4 subfield. In the cortex both mRNA types were induced, but only uPA mRNA was elevated in the retrosplenial cortex, and also in the subiculum. In the amygdaloid complex, uPA mRNA was restricted to the basolateral nucleus, whereas PAI-1 mRNA was seen throughout the structure, however, excluding this nucleus. These data show that seizure activity enhances the expression of uPA and PAI-1 genes in the brain; the patterns of enhancement suggest that the protease and its inhibitor may act in brain plasticity in synchrony, however, also independently of each other. Furthermore, the

  14. Evaluation of Prognostic Values of Tissue Plasminogen Activator and Plasminogen Activator Inhibitor-1 in Crimean-Congo Hemorrhagic Fever Patients

    PubMed Central

    Gurbuz, Yunus; Ozturk, Baris; Tutuncu, Emin Ediz; Sencan, Irfan; Cicek Senturk, Gonul; Altay, Fatma Aybala

    2015-01-01

    Background: Crimean-Congo hemorrhagic fever (CCHF) is a widespread disease in Turkey, and was responsible for many deaths in endemic regions during the last decade. The pathogenesis of the disease is not fully understood yet. Objectives: In this study we aimed to determine the levels of tissue plasminogen activator (tPA) and Plasminogen activator inhibitor-1 (PAI-1) as predictors of prognosis in CCHF. Patients and Methods: Patients who were diagnosed by the polymerase chain reaction (PCR) and IgM positivity in the reference laboratory were included in this study. Tissue Plasminogen activator and PAI-1 levels were measured by the enzyme linked immunosorbent assay (ELISA) using a commercial kit (human t-PA ELISA and human PAL-1 ELISA; BioVendor research and diagnostic products, BioVendor-Laboratorni medicina a.s., Brno, Czech Republic). Results: A total of 46 patients participated in this study. The significant differences between recovering patients and the patients who died, regarding Aspartate aminotransferase (AST), Creatine Phosphokinase (CPK), Lactate Dehydrogenase (LDH), Prothrombin Time (PT), activated Partial Thromboplastin time (aPTT), and thrombocyte and fibrinogen levels, were consistent with many clinical studies in the literature. The fatal cases were found to have higher tPA and PAI-1 levels in contrast to the patients who completely recovered. Conclusions: We think that these findings may help the progress of understanding of CCHF pathogenesis. PMID:26587219

  15. Serum Level of Plasminogen Activator Inhibitor Type-1 in Addicted Patients with Coronary Artery Disease

    PubMed Central

    Forood, Afsaneh; Malekpour-Afshar, Reza; Mahdavi, Amin

    2014-01-01

    Background Plasminogen activator inhibitor-1 (PAI-1) is a glycoprotein with inhibitory effects on the formation of plasmin from plasminogen by plasminogen activator. Thus, it prevents clot lysis in vessel walls. Several evidences prove the relationship between coronary artery disease and response to fibrinolytic therapy in patients with myocardial infarction (MI) with PAI-1 level. Opium addiction is one of the most important factors in causing MI and cardiovascular events. This is due to it causing imbalance between coagulation and anticoagulation factors in the blood. This study was designed and implemented to determine the levels of PAI-I in opium-addicted patients with coronary artery disease in comparison with non addicts. Methods In this case-control study, 160 patients with coronary heart disease (CHD), which was confirmed by angiography results, were enrolled. All of the patients had a medical history, their creatinine levels and lipid profile were evaluated, morphine urine test was performed, and after that a blood sample was taken to determine the levels of PAI-1. Thus, the 80 patients who had a positive morphine urine test result formed the case group, and the control group was constituted of the 80 patients with negative morphine test results. The two groups were matched. Findings Average level of PAI-1 in the control group was 2.4 ± 2.6 and in the case group was 8.8 ± 9.1 and it was statistically significant (P < 0.001). The frequency of two vessel disease was higher in opium addicted patients than non-addicted patients and this was statistically significant (P = 0.030). However, the frequency of single vessel and three vessel disease was the same in the two groups. The two groups had no differences in age, lipid profile, and creatinine level. Moreover, females are at a higher risk of high PAI-1 levels. Conclusion PAI-1 levels in opium addicted patients with CHD are higher than other patients. In these patients, the risk of atherosclerosis and MI is

  16. Optimized immunohistochemistry in combination with image analysis: a reliable alternative to quantitative ELISA determination of uPA and PAI-1 for routine risk group discrimination in breast cancer.

    PubMed

    Lang, D S; Heilenkötter, U; Schumm, W; Behrens, O; Simon, R; Vollmer, E; Goldmann, T

    2013-10-01

    The determination of the invasion markers urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) has further improved the possibilities for individualized therapy of breast cancer. To date, quantitative measurement by ELISA, that needs large amounts of fresh, frozen material, is the only standardized procedure for diagnostic purposes. Therefore, the aim of this study was the establishment of a reliable alternative method based on immunohistochemistry (IHC) and image analysis requiring only small amounts of fixed tumor tissue. Protein expression of uPA and PAI-1 was analyzed in HOPE-fixed tumor samples using tissue microarrays (TMAs) and semiquantitative image analysis. The results of both methods were significantly correlated and risk assessment showed an overall concordance of 78% (83/107; high- and low-risk) and of 94% (74/79) regarding only high-risk patients. The data demonstrate that optimized IHC in combination with image analysis can provide adequate clinical significance compared to ELISA-derived determination of uPA and PAI-1.

  17. Differential regulation of plasminogen activator and inhibitor gene transcription by the tumor suppressor p53.

    PubMed Central

    Kunz, C; Pebler, S; Otte, J; von der Ahe, D

    1995-01-01

    The ability of p53 to activate or repress transcription suggests that its biological function as tumor suppressor is in part accomplished by regulating a number of genes including such required for inhibition of cell growth. We here give evidence that p53 also may regulate genes responsible for the proteolytic degradation of the extracellular matrix, which is considered a crucial feature for local invasion and metastasis of neoplastic cells. An important and highly regulated cascade of such proteolytic events involves the plasminogen activator system. We show that wild-type p53 represses transcription from the enhancer and promoter of the human urokinase-type (u-PA) and the tissue-type plasminogen activator (t-PA) gene through a non-DNA binding mechanism. Oncogenic mutants lost the repressing activity. In contrast, wild-type but not mutant p53 specifically binds to and activates the promoter of the plasminogen activator inhibitor type-1 (PAI-1) gene. Interestingly, one of the p53 mutants (273his) inhibited PAI-1 promoter activity. Our results suggest that altered function of oncogenic forms of p53 may lead to altered expression of the plasminogen activators and their inhibitor(s) and thus to altered activation of the plasminogen/plasmin system during tumor progression. Images PMID:7479001

  18. Plasminogen activator inhibitor 1--insulin-like growth factor binding protein 3 cascade regulates stress-induced senescence.

    PubMed

    Elzi, David J; Lai, Yanlai; Song, Meihua; Hakala, Kevin; Weintraub, Susan T; Shiio, Yuzuru

    2012-07-24

    Cellular senescence is widely believed to play a key role in tumor suppression, but the molecular pathways that regulate senescence are only incompletely understood. By using a secretome proteomics approach, we identified insulin-like growth factor binding protein 3 (IGFBP3) as a secreted mediator of breast cancer senescence upon chemotherapeutic drug treatment. The senescence-inducing activity of IGFBP3 is inhibited by tissue-type plasminogen activator-mediated proteolysis, which is counteracted by plasminogen activator inhibitor 1 (PAI-1), another secreted mediator of senescence. We demonstrate that IGFBP3 is a critical downstream target of PAI-1-induced senescence. These results suggest a role for an extracellular cascade of secreted proteins in the regulation of cellular senescence.

  19. Transforming growth factor-1 promotes the transcriptional activation of plasminogen activator inhibitor type 1 in carcinoma-associated fibroblasts.

    PubMed

    Zhu, Yu; Yin, Wan-Le; Ba, Yu-Feng; Tian, Lin; Gu, Zhi-Qiang; Zhang, Ming-Sheng; Zhong, Chu-Nan

    2012-11-01

    Carcinoma-associated fibroblasts (CAFs) play a pivotal role in promoting the growth, invasion and metastasis of tumor cells. However, to date little is known about the oncogenic mechanisms of CAFs. This study aimed to identify the microenvironmental factors involved in tumor development and progression directed by CAFs in liver metastases. Tissue samples collected from 20 patients with colorectal liver metastases were used in this study. Histological and morphological characterization of the samples was performed using hybridization and immunohistological assays. The mRNA expression of α-smooth muscle actin (α-SMA) was measured by northern blotting. The expression of plasminogen activator inhibitor type 1 (PAI-1) was measured by enzyme-linked immunosorbent assay (ELISA). As a result, co-expression of Thy-1 (CD90) and α-SMA was identified in CAFs, while normal liver samples were negative for α-SMA and Thy-1. Compared with epidermal growth factor (EGF) and tumor necrosis factor (TNF) incubation, the expression of α-SMA increased significantly following transforming growth factor-1 (TGF-1) incubation (P<0.05), while platelet-derived growth factor (PDGF) caused a significant suppression of α-SMA expression (P<0.05). PAI-1 expression was significantly lower in unstimulated fibroblasts compared to TGF-1-treated fibroblasts (P<0.01). The levels of PAI-1 transcription were significantly higher in CAFs from the patient samples compared with the healthy controls. Taken together, our findings suggest that CAFs may be important in migration, matrix degradation, invasion and angiogenesis of tumors, and TGF-1 may promote the activation of PAI-1 transcription in CAFs.

  20. Regulatory role of microRNA-30b and plasminogen activator inhibitor-1 in the pathogenesis of cognitive impairment

    PubMed Central

    LI, XIUQIN; GAO, YONG; MENG, ZHAOYUN; ZHANG, CUI; QI, QINDE

    2016-01-01

    The present study aimed to investigate the role of plasminogen activator inhibitor-1 (PAI-1) in drug-induced early cognitive impairment and the underlying mechanism concerning microRNA (miR)-30b. A mouse model of cognitive impairment was established by intraperitoneal injection of scopolamine (2 mg/kg body weight) for 13 days. Behavioral performance was assessed using the Morris water maze (MWM) test. The mRNA expression levels of PAI-1 and miR-30b were detected using quantitative polymerase chain reaction (qPCR). The protein expression levels of PAI-1 in the hippocampus and blood were determined using western blot analysis and enzyme-linked immunosorbent assays. The MWM test demonstrated that, on days 3 and 4, the escape latency was significantly elevated in the model mice in comparison with control group (P<0.05). In addition, the length of swimming path was significantly increased (P<0.05), while the number of times of crossing the platform location was significantly reduced in the model mouse group (P<0.05) in comparison with the control group. qPCR demonstrated that the mRNA expression levels of PAI-1 in the model mice was significantly elevated in the hippocampus and blood in comparison with the control group (P<0.01). Furthermore, western blot analysis and enzyme-linked immunosorbent assay demonstrated that the protein expression levels of PAI-1 were significantly elevated in the hippocampus and blood in the model group, in comparison with the control group (P<0.05). Notably, the levels of miR-30b in the hippocampus and blood were significantly decreased in the model mice in comparison with the control group (P<0.01). To conclude, the expression levels of PAI-1 were significantly elevated in mice with scopolamine-induced cognitive impairment, which may be associated with the downregulation of miR-30b. The findings from the present study suggest that miR-30b may be involved in the regulation of PAI-1, which would contribute to the pathogenesis of cognitive

  1. Leptin links with plasminogen activator inhibitor-1 in human obesity: the SABPA study.

    PubMed

    Pieterse, Chiné; Schutte, Rudolph; Schutte, Aletta E

    2015-07-01

    The relationship between obesity and the development of cardiovascular disease is well established. However, the underlying mechanisms contributing to vascular disease and increased cardiovascular risk in the obese remain largely unexplored. Since leptin exerts direct vascular effects, we investigated leptin and the relationship thereof with circulating markers of vascular damage, namely plasminogen activator inhibitor-1 antigen (PAI-1(ag)), von Willebrand factor antigen (vWF(ag)) and urinary albumin-to-creatinine ratio (ACR). The study included a bi-ethnic population of 409 African and Caucasian teachers who were stratified into lean (<0.5) and obese (⩾0.5) groups according to waist-to-height ratio. We obtained ambulatory blood pressure measurements and determined serum leptin levels, PAI-1(ag), vWF(ag) and ACR, as markers of vascular damage. The obese group had higher leptin (P<0.001) and PAI-1(ag) (P<0.001) levels and a tendency existed for higher vWF(ag) (P=0.068). ACR did not differ between the two groups (P=0.21). In single regression analyses positive associations existed between leptin and all markers of vascular damage (all P<0.001) only in the obese group. After adjusting for covariates and confounders in multiple regression analyses, only the association between leptin and PAI-1(ag) remained (R(2)=0.440; β=0.293; P=0.0021). After adjusting for gender, ethnicity and age, additional analyses indicated that leptin also associated with fibrinogen and clot lysis time in both lean and obese groups, which in turn is associated with 24- h blood pressure and pulse pressure. This result provides evidence that elevated circulating leptin may directly contribute to vascular damage, possibly through mechanism related to thrombotic vascular disease.

  2. Leptin links with plasminogen activator inhibitor-1 in human obesity: the SABPA study.

    PubMed

    Pieterse, Chiné; Schutte, Rudolph; Schutte, Aletta E

    2015-07-01

    The relationship between obesity and the development of cardiovascular disease is well established. However, the underlying mechanisms contributing to vascular disease and increased cardiovascular risk in the obese remain largely unexplored. Since leptin exerts direct vascular effects, we investigated leptin and the relationship thereof with circulating markers of vascular damage, namely plasminogen activator inhibitor-1 antigen (PAI-1(ag)), von Willebrand factor antigen (vWF(ag)) and urinary albumin-to-creatinine ratio (ACR). The study included a bi-ethnic population of 409 African and Caucasian teachers who were stratified into lean (<0.5) and obese (⩾0.5) groups according to waist-to-height ratio. We obtained ambulatory blood pressure measurements and determined serum leptin levels, PAI-1(ag), vWF(ag) and ACR, as markers of vascular damage. The obese group had higher leptin (P<0.001) and PAI-1(ag) (P<0.001) levels and a tendency existed for higher vWF(ag) (P=0.068). ACR did not differ between the two groups (P=0.21). In single regression analyses positive associations existed between leptin and all markers of vascular damage (all P<0.001) only in the obese group. After adjusting for covariates and confounders in multiple regression analyses, only the association between leptin and PAI-1(ag) remained (R(2)=0.440; β=0.293; P=0.0021). After adjusting for gender, ethnicity and age, additional analyses indicated that leptin also associated with fibrinogen and clot lysis time in both lean and obese groups, which in turn is associated with 24- h blood pressure and pulse pressure. This result provides evidence that elevated circulating leptin may directly contribute to vascular damage, possibly through mechanism related to thrombotic vascular disease. PMID:25740294

  3. PAI-1-regulated miR-21 defines a novel age-associated fibrogenic pathway in muscular dystrophy.

    PubMed

    Ardite, Esther; Perdiguero, Eusebio; Vidal, Berta; Gutarra, Susana; Serrano, Antonio L; Muñoz-Cánoves, Pura

    2012-01-01

    Disruption of skeletal muscle homeostasis by substitution with fibrotic tissue constitutes the principal cause of death in Duchenne muscular dystrophy (DMD) patients, yet the implicated fibrogenic mechanisms remain poorly understood. This study identifies the extracellular PAI-1/urokinase-type plasminogen activator (uPA) balance as an important regulator of microribonucleic acid (miR)-21 biogenesis, controlling age-associated muscle fibrosis and dystrophy progression. Genetic loss of PAI-1 in mdx dystrophic mice anticipated muscle fibrosis through these sequential mechanisms: the alteration of collagen metabolism by uPA-mediated proteolytic processing of transforming growth factor (TGF)-β in muscle fibroblasts and the activation of miR-21 expression, which inhibited phosphatase and tensin homologue and enhanced AKT signaling, thus endowing TGF-β with a remarkable cell proliferation-promoting potential. Age-associated fibrogenesis and muscle deterioration in mdx mice, as well as exacerbated dystrophy in young PAI-1(-/-) mdx mice, could be reversed by miR-21 or uPA-selective interference, whereas forced miR-21 overexpression aggravated disease severity. The PAI-1-miR-21 fibrogenic axis also appeared dysregulated in muscle of DMD patients, providing a basis for effectively targeting fibrosis and muscular dystrophies in currently untreatable individuals.

  4. The mechanism and significance of synergistic induction of the expression of plasminogen activator inhibitor-1 by glucocorticoid and transforming growth factor beta in human ovarian cancer cells.

    PubMed

    Pan, Xiao-yu; Wang, Yan; Su, Jie; Huang, Gao-xiang; Cao, Dong-mei; Qu, Shen; Lu, Jian

    2015-05-15

    Plasminogen activator inhibitor-1 (PAI-1) plays a key role in tissue remodeling and tumor development by suppression of plasminogen activator function. Glucocorticoids (GCs) and transforming growth factor beta (TGF-β) signal pathways cross-talk to regulate gene expression, but the mechanism is poorly understood. Here we investigated the mechanism and significance of co-regulation of PAI-1 by TGF-β and dexamethasone (DEX), a synthetic glucocorticoid in ovarian cancer cells. We found that TGF-β and DEX showed rapidly synergistic induction of PAI-1 expression, which contributed to the early pro-adhesion effects. The synergistic induction effect was accomplished by several signal pathways, including GC receptor (GR) pathway and TGF-β-activated p38MAPK, ERK and Smad3 pathways. TGF-β-activated p38MAPK and ERK pathways cross-talked with GR pathway to augment the expression of PAI-1 through enhancing DEX-induced GR phosphorylation at Ser211 in ovarian cancer cells. These findings reveal possible novel mechanisms by which TGF-β pathways cooperatively cross-talk with GR pathway to regulate gene expression.

  5. Induction of oxidative stress and inhibition of plasminogen activator inhibitor-1 production in endothelial cells following exposure to organic extracts of diesel exhaust particles and urban fine particles.

    PubMed

    Furuyama, Akiko; Hirano, Seishiro; Koike, Eiko; Kobayashi, Takahiro

    2006-03-01

    Endothelial cells play important roles in anticoagulant and fibrinolytic systems. Recent studies suggest that increases in ambient particulate matter (PM) levels have been associated with an increase in mortality rate from cardiovascular diseases. We examined the production of heme oxygenase-1 (HO-1) and factors related to the fibrinolytic function by rat heart microvessel endothelial cells exposed to organic extracts of diesel exhaust particles (OE-DEP) and urban fine particles (OE-UFP) to investigate the direct effects of these soluble organic fractions in these PM on the fibrinolytic function of endothelial cells. The cell monolayer exposed to 10 microg/ml OE-DEP produced a larger amount of HO-1 than cells exposed to 10 microg/ml OE-UFP. OE-DEP and OE-UFP exposure reduced plasminogen activator inhibitor-1 (PAI-1) production by the cells but did not affect the production of thrombomodulin, tissue-type plasminogen activator, or urokinase-type plasminogen activator. Increased PAI-1 synthesis in response to treatment with 1.0 ng/ml tumor necrosis factor-alpha or 0.5 ng/ml transforming growth factor-beta1 was reduced by OE-DEP exposure. Suppression of PAI-1 production by OE-DEP exposure was mediated through oxidative stress and was independent of HO-1 activity. These results suggest that exposure to the soluble organic fraction of PM and DEP induced oxidative stress and reduced the PAI-1 production of endothelial cells.

  6. Targeting of plasminogen activator inhibitor 1 improves fibrinolytic therapy for tetracycline-induced pleural injury in rabbits.

    PubMed

    Florova, Galina; Azghani, Ali; Karandashova, Sophia; Schaefer, Chris; Koenig, Kathleen; Stewart-Evans, Kris; Declerck, Paul J; Idell, Steven; Komissarov, Andrey A

    2015-04-01

    Endogenous active plasminogen activator inhibitor 1 (PAI-1) was targeted in vivo with monoclonal antibodies (mAbs) that redirect its reaction with proteinases to the substrate branch. mAbs were used as an adjunct to prourokinase (single-chain [sc] urokinase [uPA]) intrapleural fibrinolytic therapy (IPFT) of tetracycline-induced pleural injury in rabbits. Outcomes of scuPA IPFT (0.25 or 0.0625 mg/kg) with 0.5 mg/kg of mouse IgG or mAbs (MA-33H1F7 and MA-8H9D4) were assessed at 24 hours. Pleural fluid (PF) was collected at 0, 10, 20, and 40 minutes and 24 hours after IPFT and analyzed for plasminogen activating (PA), uPA, fibrinolytic activities, levels of total plasmin/plasminogen, α-macroglobulin (αM), mAbs/IgG antigens, free active uPA, and αM/uPA complexes. Anti-PAI-1 mAbs, but not mouse IgG, delivered with an eightfold reduction in the minimal effective dose of scuPA (from 0.5 to 0.0625 mg/kg), improved the outcome of IPFT (P < 0.05). mAbs and IgG were detectable in PFs at 24 hours. Compared with identical doses of scuPA alone or with IgG, treatment with scuPA and anti-PAI-1 mAbs generated higher PF uPA amidolytic and PA activities, faster formation of αM/uPA complexes, and slower uPA inactivation. However, PAI-1 targeting did not significantly affect intrapleural fibrinolytic activity or levels of total plasmin/plasminogen and αM antigens. Targeting PAI-1 did not induce bleeding, and rendered otherwise ineffective doses of scuPA able to improve outcomes in tetracycline-induced pleural injury. PAI-1-neutralizing mAbs improved IPFT by increasing the durability of intrapleural PA activity. These results suggest a novel, well-tolerated IPFT strategy that is tractable for clinical development.

  7. Portal Vein Thrombosis in a Preterm Newborn with Mutation of the MTHFR and PAI-1 Genes and Sepsis by Candida parapsilosis.

    PubMed

    Giuffrè, Mario; Verso, Clelia Lo; Serra, Gregorio; Moceri, Giovanni; Cimador, Marcello; Corsello, Giovanni

    2016-09-01

    Objective This report discusses the role of both congenital and acquired risk factors in the pathogenesis of portal vein thrombosis (PVT). Study Design We describe the clinical management and treatment of PVT in a preterm newborn with a homozygous mutation of the methylenetetrahydrofolate reductase (MTHFR) and plasminogen activator inhibitor-1 (PAI-1) genes and sepsis by Candida parapsilosis. Results Although literature data suggest a minor role of genetic factors in thrombophilia in the case of only one mutation, we hypothesize that combined thrombophilic genetic defects may have a cumulative effect and significantly increase the thrombotic risk. Conclusion It could be appropriate to include more detailed analyses of procoagulant and fibrinolytic factors in the diagnostic workup of neonatal thrombosis, also through the investigation of genetic polymorphisms. The anticoagulant therapy and the removal of concurrent risk factors remain basic steps for the adequate management and prevention of complications. PMID:27603544

  8. Tumor-biological factors uPA and PAI-1 as stratification criteria of a multicenter adjuvant chemotherapy trial in node-negative breast cancer.

    PubMed

    Prechtl, A; Harbeck, N; Thomssen, C; Meisner, C; Braun, M; Untch, M; Wieland, M; Lisboa, B; Cufer, T; Graeff, H; Selbmann, K; Schmitt, M; Jänicke, F

    2000-01-01

    In axillary node-negative primary breast cancer, 70% of the patients will be cured by locoregional treatment alone. Therefore, adjuvant systemic therapy is only needed for those 30% of node-negative patients who will relapse after primary therapy and eventually die of metastases. Traditional histomorphological and clinical factors do not provide sufficient information to allow accurate risk group assessment in order to identify node-negative patients who might benefit from adjuvant systemic therapy. In the last decade various groups have reported a strong and statistically independent prognostic impact of the serine protease uPA (urokinase-type plasminogen activator) and its inhibitor PAI-1 (plasminogen activator inhibitor type 1) in node-negative breast cancer patients. Based on these data, a prospective multicenter therapy trial in node-negative breast cancer patients was started in Germany in June 1993, supported by the German Research Association (DFG). Axillary node-negative breast cancer patients with high levels of either or both proteolytic factors in the tumor tissue were randomized to adjuvant CMF chemotherapy versus observation only. Recruitment was continued until the end of 1998, by which time 684 patients had been enrolled. Since then, patients have been followed up in order to assess the value of uPA and PAI-1 determination as an adequate selection criterion for adjuvant chemotherapy in node-negative breast cancer patients. This paper reports on the rationale and design of this prospective multicenter clinical trial, which may have an impact on future policies in prognosis-oriented treatment strategies.

  9. Heparanase procoagulant activity, factor Xa, and plasminogen activator inhibitor 1 are increased in shift work female nurses.

    PubMed

    Nadir, Yona; Saharov, Gleb; Hoffman, Ron; Keren-Politansky, Anat; Tzoran, Inna; Brenner, Benjamin; Shochat, Tamar

    2015-07-01

    Epidemiologic studies indicate on an increased risk of cardiovascular disease and cancer in shift workers, although the underlying mechanism is obscure. Heparanase directly enhances tissue factor (TF) activity leading to increased factor Xa production and subsequent activation of the coagulation system. In the present study, a comparison of coagulation markers among healthy shift working (SW) vs. healthy daytime working (DW) female nurses was performed. Thirty SW and 30 DW female nurses were enrolled. For each of the 60 participants, blood was drawn between 7:00 and 8:00 a.m. and at least 8 h after the last work shift. Plasma was studied for coagulation marker that included TF/heparanase procoagulant activity, TF activity, heparanase procoagulant activity, heparanase level, factor Xa level, plasminogen activator inhibitor 1 (PAI-1), plasminogen, α2-antiplasmin, fibrinogen, global protein C, von Willebrand factor, and D-dimer by chromogenic assays and enzyme-linked immunosorbent assays (ELISAs). Sleep quality was assessed by self-report according to the Pittsburgh Sleep Quality Index. The heparanase procoagulant activity increased by 2-fold and the TF/heparanase procoagulant activity increased by 1.5-fold in SW nurses compared to DW nurses (P < 0.05). Factor Xa levels and PAI-1 levels were significantly higher among SW nurses compared to the DW group (22 vs. 18 ng/ml, P < 0.05, and 32 vs. 22 ng/ml, P < 0.005, respectively). No significant differences were found in the other tested coagulation markers between the study groups. Heparanase procoagulant activity, factor Xa level, and PAI-1 level were significantly higher in SW nurses compared to the DW group. These alterations of blood coagulation activation may potentially contribute to cardiovascular and cancer morbidity.

  10. Impacts of aging and amyloid-β deposition on plasminogen activators and plasminogen activator inhibitor-1 in the Tg2576 mouse model of Alzheimer's disease.

    PubMed

    Bi Oh, Shin; Suh, Nayoung; Kim, Inki; Lee, Joo-Yong

    2015-02-01

    Plasminogen activators (PAs), which convert plasminogen into the fibrinolytic protease plasmin, may initiate the degradation of amyloid-β (Aβ) to suppress the amyloid pathogenesis. In that way, tissue plasminogen activator (tPA)-mediated plasmin activation could maintain a low level of Aβ deposition to delay the pathogenesis of Alzheimer's disease (AD). In a previous study, we reported that tPA/plasmin proteolytic activity is attenuated throughout the brain during aging or with Aβ accumulation but clustered intense around the amyloid plaques in AD brain. The present study demonstrates that the altered proteolytic activity primarily results from the competition between the expressions of tPA and plasminogen activator inhibitor-1 (PAI-1) in the brains of Tg2576 Aβ-transgenic mice, as revealed by immunohistochemistry and immunoblot assays. Compared with that in the brains of younger Tg2576 mice, tPA protein is generally reduced throughout the brain in older Tg2576 mice but elevated near amyloid plaques. In contrary, PAI-1 expression increases during aging or Aβ deposition with its clusters surrounding amyloid plaques. No significant alteration in the expression of urokinase plasminogen activator (uPA) is detected. These results suggest reciprocal feedback influences between tPA, PAI-1 and Aβ during aging and amyloid pathogenesis in AD brain; tPA-mediated plasmin activity is declined throughout the brain causing Aβ deposition during aging, and the Aβ deposits locally attract the cluster of tPA and/or PAI-1 around their deposits to competitively determine tPA/plasmin-mediated Aβ proteolysis.

  11. Comparison of plasminogen activator inhibitor-1 concentration in insulin-resistant versus insulin-sensitive healthy women.

    PubMed

    Abbasi, F; McLaughlin, T; Lamendola, C; Lipinska, I; Tofler, G; Reaven, G M

    1999-11-01

    The primary goal of this investigation was to see whether plasminogen activator inhibitor-1 (PAI-1) concentrations varied as a function of differences in insulin-mediated glucose disposal in 2 groups of healthy women matched for every other variable that might play a role in regulation of PAI-1. For this purpose, we recruited 32 healthy women, divided on the basis of their steady-state plasma glucose (SSPG) concentrations during the insulin suppression test into an insulin-resistant (SSPG=216+/-12 mg/dL, n=16) and an insulin-sensitive (94+/-6 mg/dL, n=16) group. PAI-1 antigen concentrations were significantly higher (26+/-4 versus 14+/-3 ng/mL, P<0.02) in the insulin-resistant group. In addition, fasting plasma insulin (18+/-3 versus 11+/-2 microU/mL, P<0.02) and triglyceride (160+/-19 versus 93+/-10 mg/dL, P<0.001) concentrations were higher in the insulin-resistant individuals, whereas HDL concentrations were lower (44+/-3 versus 58+/-3 mg/dL, P<0.005). However, the 2 groups were essentially identical in terms of age, menopausal status, hormone replacement therapy, body mass index (BMI), ratio of waist-to-hip girth, and blood pressure. When the experimental population was considered as 1 group, there were statistically significant correlations between PAI-1 antigen and the following variables: adjusting for differences in age and BMI, SSPG (r=0.56, P<0.001); triglyceride (r=0.39, P<0.05); and HDL cholesterol (r=-0. 65, P<0.001) concentrations. Finally, multiple regression analysis revealed the major determinants of PAI-1 to be insulin resistance, or insulin concentration, and HDL cholesterol. These results: 1) demonstrate that PAI-1 concentrations are higher in healthy, insulin-resistant women as compared with insulin-sensitive individuals, independent of differences in BMI or ratio of waist-to-hip girth; and 2) provide another mechanism by which insulin-resistant individuals are at increased thrombotic cardiovascular risk.

  12. Application of Long-Acting VLHL PAI-1 during Sutureless Partial Nephrectomy in Mice Reduces Bleeding

    PubMed Central

    Shahrour, Khaled; Keck, Rick; Jankun, Jerzy

    2015-01-01

    PAI-1 prevents lysis of blood clot by inhibiting the urokinase and tPA induced conversion of plasminogen to plasmin. VLHL PAI-1 protein mutant was created to extend half-life over 700 hours. The objective of this paper was to test VLHL PAI-1 effects on bleeding during partial nephrectomy in mice. All animals had a left partial nephrectomy after intravenous infusion of saline or tPA. The animals were divided into four groups. Group 1 was infused with saline and kidney was exposed to saline too; Group 2 was infused with saline and kidney was exposed to PAI-1. Group 3 was infused with tPA and kidney was exposed to saline, while Group 4 was infused with tPA and kidney was exposed to PAI-1. Preweighed gauze containing PAI-1 or saline was then applied to the kidney for 30 minutes. The gauze was afterward weighed and blood loss was measured by subtracting the preweight of gauze from the final weight. We have observed a statistically significant (P ≤ 0.05) reduction of bleeding in PAI-1-treated group in comparison to saline and tPA-treated groups. Based on these results we propose that VLHL PAI-1 can be used therapeutically in limiting the flow of blood from renal wounds. PMID:25883959

  13. Effects of a diet containing Brazilian propolis on lipopolysaccharide-induced increases in plasma plasminogen activator inhibitor-1 levels in mice

    PubMed Central

    Ohkura, Naoki; Oishi, Katsutaka; Kihara-Negishi, Fumiko; Atsumi, Gen-ichi; Tatefuji, Tomoki

    2016-01-01

    Background: Brazilian propolis has many biological activities including the ability to help prevent thrombotic diseases, but this particular effect has not been proven. Plasma levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, increase under inflammatory conditions such as infection, obesity and atherosclerosis and such elevated levels predispose individuals to a risk of developing thrombotic diseases. Aim: This study aimed to determine the effects of a diet containing Brazilian propolis on lipopolysaccharide (LPS)-induced increases in plasma PAI-1 levels. Materials and Methods: Mice were fed with a diet containing 0.5% (w/w) Brazilian propolis for 8 weeks. Thereafter, the mice were subcutaneously injected with saline containing 0.015 mg/kg of LPS and sacrificed 4 h later. Results: Orally administered Brazilian propolis significantly suppressed the LPS-induced increase in PAI-1 antigen and its activity in mouse plasma. Conclusion: This study indicated that Brazilian propolis contains natural products that can decrease thrombotic tendencies in mice. PMID:27757277

  14. Polymorphism in methylenetetrahydrofolate reductase, plasminogen activator inhibitor-1, and apolipoprotein E in hemodialysis patients.

    PubMed

    Al-Muhanna, Fahad; Al-Mueilo, Samir; Al-Ali, Amein; Larbi, Emmanuel; Rubaish, Abdullah; Abdulmohsen, Mohammed Fakhry; Al-Zahrani, Alhussain; Al-Ateeq, Suad

    2008-11-01

    The methylenetetrahydrofolate reductase (MTHFR) gene polymorphism, apolipoprotein E (apo epsilon4) gene polymorphism and polymorphism of plasminogen activator inhibitor-1 (PAI-1) have been shown to be associated with end-stage renal disease (ESRD). To determine the prevalence of these mutations in Saudi patients with ESRD on hemodialysis, we studied the allelic frequency and genotype distribution in patients receiving hemodialysis and in a control group, all residing in the Eastern Province of Saudi Arabia. The genotypes were determined using allele specific hybridization procedures and were confirmed by restriction fragment length polymorphism. The T allele frequency and homozygous genotype of MTHFR in ESRD patients were 14% and 2.4%, respectively compared to 13.4% and 0%, respectively in the control group. The allele frequency and homozygous genotype of 4G/4G PAI-1 gene polymorphism were 46.4% and 4.8% respectively in ESRD patients compared to 57.1% and 32% respectively in the control group. The apo s4 allele frequency and homozygous genotype distribution in hemodialysis patients were 7% and 2.4%, respectively compared to 13% and 2% in the control group. Although allele frequency of C677T of MTHFR was statistically similar in the hemodialysis patients and in the control group, the homozygotes T allele genotype was over represented in the hemodialysis group compared to normal. The prevalence of PAI-1 4G/4G polymorphism in ESRD patients was lower when compared to the control group. The prevalence of apo s4 allele did not differ significantly between the two groups. The present results demonstrate that all three studied polymorphic mutations are present in our population and that they may contribute to the etiology of the disease in our area. PMID:18974580

  15. Angiotensinogen and Plasminogen Activator Inhibitor-1 Gene Polymorphism in Relation to Renovascular Disease

    SciTech Connect

    Reis, Kadriye Altok Onal, Baran; Gonen, Sevim; Arinsoy, Turgay; Erten, Yasemin; Ilgit, Erhan; Soylemezoglu, Oguz; Derici, Ulver; Guz, Galip; Bali, Musa; Sindel, Sukru

    2006-02-15

    The present study was designed to evaluate angiotensinogen (AGT) M235T and plasminogen activator inhibitor-1 (PAI-1) (4G/5G) polymorphisims in relation to the occurrence of atherosclerotic renal artery stenosis (ARAS) and recurrent stenosis. In this study, 30 patients were enrolled after angiographic demonstration of ARAS; 100 healthy subjects for AGT polymorphism and 80 healthy subjects for PAI-1 polymorphism were considered the control group. The patients were followed for a mean 46.1 {+-} 9.2 months. The patients had significantly higher frequencies of the MT genotype and the T allele than control group ({chi}{sup 2} = 18.2, p < 0.001 and {chi}{sup 2} = 11.5 p < 0.001). There were no significant differences in the PAI-1 genotype and allele findings when the data for all patients were compared with that for the controls ({chi}{sup 2}= 2.45, p = 0.29 and {chi}{sup 2} = 0.019, p = 0.89). There were no significant differences in the genotype and allele findings for the patients with and without restenosis (p > 0.05). The C-reactive protein (CRP) level was higher in the patients with restenosis than in the patients without restenosis (7.694 {+-} 0.39 mg/L and 1.56 {+-} 1.08 mg/L) (p = 0.001). Our results suggest that the M235T MT genotype and T allele might be associated with increased risk of atherosclerotic renal artery stenosis. The CRP level might be an independent predictor for recurrent stenosis.

  16. Inhibitory effects of C-type natriuretic peptide on the differentiation of cardiac fibroblasts, and secretion of monocyte chemoattractant protein-1 and plasminogen activator inhibitor-1.

    PubMed

    Li, Zhi-Qiang; Liu, Ying-Long; Li, Gang; Li, Bin; Liu, Yang; Li, Xiao-Feng; Liu, Ai-Jun

    2015-01-01

    The present study aimed to investigate the effect of C-type natriuretic peptide (CNP) on the function of cardiac fibroblasts (CFs). Western blotting was used to investigate the expression of myofibroblast marker proteins: α-smooth muscle actin (α-SMA), extra domain-A fibronectin, collagen I and collagen III, and the activity of extracellular signal-regulated kinase 1/2 (ERK1/2). Immunofluorescence was used to examine the morphological changes; a transwell assay was used to analyze migration, and reverse transcription-quantitative polymerase chain reaction and ELISA were employed to determine the mRNA expression and protein secretion of monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1). The results demonstrated that CNP significantly reduced the protein expression of α-SMA, fibronectin, collagen I and collagen III, and suppressed the migratory ability of CFs. Additionally, the mRNA and protein expression of MCP-1 and PAI-1 was inhibited under the CNP treatment; and this effect was mediated by the inhibition of the ERK1/2 activity. In conclusion, CNP inhibited cardiac fibroblast differentiation and migration, and reduced the secretion of MCP-1 and PAI-1, which demonstrates novel mechanisms to explain the antifibrotic effect of CNP.

  17. A suppressive effect of prostaglandin E2 on the expression of SERPINE1/plasminogen activator inhibitor-1 in human articular chondrocytes: An in vitro pilot study

    PubMed Central

    Masuko, Kayo; Murata, Minako; Suematsu, Naoya; Okamoto, Kazuki; Yudoh, Kazuo; Shimizu, Hiroyuki; Beppu, Moroe; Nakamura, Hiroshi; Kato, Tomohiro

    2009-01-01

    Prostaglandin E2 (PGE2) is expressed in articular joints with inflammatory arthropathy and may exert catabolic effects leading to cartilage degradation. As we observed in a preliminary experiment that PGE2 suppressed the expression of SERPINE1/plasminogen activator inhibitor (PAI)-1 mRNA in chondrocytes, we focused on the effect of PGE2 on PAI-1 in a panel of cultured chondrocytes obtained from osteoarthritic patients. Specifically, articular cartilage specimens were obtained from patients with osteoarthritis who underwent joint surgery. Isolated chondrocytes were cultured in vitro as a monolayer and stimulated with PGE2. Stimulated cells and culture supernatants were analyzed using Western blotting and enzyme-linked immunosorbent assay. The results confirmed that the in vitro PGE2 stimulation suppressed the expression of PAI-1 in the tested chondrocyte samples. The inhibitory effect was partly abrogated by an antagonist of EP4 receptor of PGE2, but not by an EP2 antagonist. Although PGE2 induced activations of mitogen-activated protein kinases (MAPK), blocking of the MAPK did not abrogate the suppressive effect of PGE2, implying a distinct signaling pathway. In summary, prostaglandin is suggested to modulate the plasminogen system in chondrocytes. Further elucidation of the interaction might open a new avenue to understand the degradative process of cartilage.

  18. PAI-1 4G-4G and MTHFR 677TT in non-hepatitis C virus/hepatitis B virus-related liver cirrhosis

    PubMed Central

    Pasta, Linda; Pasta, Francesca

    2015-01-01

    the inflammation response, and causing the hepatic fibrosis and augmented intrahepatic vascular resistance typical of LC. PAI-1 4G-4G and MTHFR 677TT screening of LC patients could be useful, mainly in those with NVLC, to identify patients in which new drug therapies based on the attenuation of the hepatic stellate cells activation or other mechanisms could be more easily evaluated. PMID:26689658

  19. Prevalence of coagulation factor XIII and plasminogen activator inhibitor-1 gene polymorphisms among Egyptian women suffering from unexplained primary recurrent miscarriage.

    PubMed

    Elmahgoub, Iman Rifaat; Afify, Reham Abdelaleem; Abdel Aal, Asmaa Ahmed; El-Sherbiny, Walid Sayed

    2014-06-01

    Recurrent miscarriage (RM) is an obstetric challenge. Polymorphisms of factor XIII (FXIII) and plasminogen activator inhibitor-1 (PAI-1) may cause an imbalance between coagulation and fibrinolysis that can end in RM. The aim of the work was to determine the prevalence of FXIII Val34Leu and PAI-1 4G/5G gene polymorphisms in Egyptian women presenting with unexplained primary first trimester RM. Genotyping of 120 unexplained primary first trimester RM patients and 130 healthy controls by polymerase chain reaction (PCR) amplification of target genes followed by the allele-specific restriction enzyme digestion (RFLP technique). Among the cases, 67.5% of individuals had wild-type FXIII; 21.7% were heterozygous and 10.8% were homozygous for the FXIII Val34Leu polymorphism. Among controls, the proportions were 89.2%, 8.5% and 2.3% respectively. In addition, comparison between the two groups regarding Leu and 4G allele frequencies showed statistically significant differences (P values=0.0001 and 0.027 respectively). RM is more frequent in women with combined polymorphisms than in women with a single gene polymorphism (RR=3.91; OR=4.51; 95% CI=1.79-11.38; P=0.002). FXIII Val34Leu and PAI-1 4G/5G polymorphisms are prevalent in Egyptian women, with unexplained primary first trimester RM and combined polymorphisms statistically increasing the risk.

  20. The transcription factor EGR-1 suppresses transformation of human fibrosarcoma HT1080 cells by coordinated induction of transforming growth factor-beta1, fibronectin, and plasminogen activator inhibitor-1.

    PubMed

    Liu, C; Yao, J; de Belle, I; Huang, R P; Adamson, E; Mercola, D

    1999-02-12

    Re-expression of EGR-1 in fibrosarcoma HT1080 suppresses transformation including tumorigenicity (Huang, R.-P., Liu, C., Fan, Y., Mercola, D., and Adamson, E. (1995) Cancer Res. 55, 5054-5062) owing in part to up-regulation of the transforming growth factor (TGF)-beta1 promoter by EGR-1 which suppresses growth by an autocrine mechanism (Liu, C., Adamson, E., and Mercola, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11831-11836). Here we show that enhanced cell attachment contributes to the suppression via increased secretion of fibronectin (FN) and also of plasminogen activator inhibitor-1 (PAI-1). The secretion of FN and PAI-1 is strongly correlated with EGR-1 expression (RPEARSON = 0.971 and 0. 985, respectively). Addition of authentic TGF-beta1 to parental cells greatly stimulated secretion of PAI-1 but not FN, whereas addition of TGF-beta antibody or lipofection with specific antisense TGF-beta1 oligonucleotides to EGR-1-regulated cells completely inhibits the secretion of PAI-1 but not FN. However, in gel mobility shift assays pure EGR-1 or nuclear extracts of EGR-1-regulated cells specifically bind to two GC-rich elements of the human FN promoter at positions -75/-52 and -4/+18, indicating that the increased secretion of FN is likely due to direct up-regulation by EGR-1. Moreover, adhesion was greatly enhanced in EGR-1-regulated cells and was reversed by treatment with Arg-Gly-Asp (RGD) or PAI-1 antibody indicating that the secreted proteins are functional. We conclude that EGR-1 regulates the coordinated expression of gene products important for cell attachment ("oikis" factor) and normal growth control.

  1. Circulating Levels of PAI-1 and SERPINE1 4G/4G Polymorphism Are Predictive of Poor Prognosis in HCC Patients Undergoing TACE1

    PubMed Central

    Divella, Rosa; Daniele, Antonella; Abbate, Ines; Savino, Eufemia; Casamassima, Porzia; Sciortino, Giancarlo; Simone, Giovanni; Gadaleta-Caldarola, Gennaro; Fazio, Vito; Gadaleta, Cosimo Damiano; Sabbà, Carlo; Mazzocca, Antonio

    2015-01-01

    Although several molecular markers have been proposed as prognostic of disease progression in Hepatocellular carcinoma (HCC), predictive markers of response to treatment are still unsatisfactory. Here, we propose a genetic polymorphism as a potential predictive factor of poor prognosis in HCC patients treated with transcatheter arterial chemoembolization (TACE). In particular, we show that the guanosine insertion/deletion polymorphism in the promoter region of SERPINE1 gene at the − 675 bp position, named 4G/4G, predicts poor prognosis in a cohort of 75 patients with HCC undergoing TACE. By a combination of ELISA and SERPINE1 promoter study, we found that the presence of elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) in patients with 4G/4G genotype is significantly associated with reduced overall survival compared to patients with 5G/5G or 4G/5G genotype in HCC patients after TACE. Our analysis provided evidence that variation in SERPINE1 gene plays a role in defining the outcome in patients treated with TACE. In addition to a poor disease outcome, the 4G/4G variant represents an unfavorable predictive factor for response to chemotherapy as well. PMID:26310373

  2. 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

    2016-01-01

    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.

  3. 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 Central

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

    2016-01-01

    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. PMID:27092498

  4. 5-Hydroxytryptamine 2A receptor signaling cascade modulates adiponectin and plasminogen activator inhibitor 1 expression in adipose tissue.

    PubMed

    Uchida-Kitajima, Shoko; Yamauchi, Toshimasa; Takashina, Youko; Okada-Iwabu, Miki; Iwabu, Masato; Ueki, Kohjiro; Kadowaki, Takashi

    2008-09-01

    Knowledge of the regulatory factors associated with down-regulation of adiponectin gene expression and up-regulation of PAI-1 gene expression is crucial to understand the pathophysiological basis of obesity and metabolic diseases, and could establish new treatment strategies for these conditions. We showed that expression of 5-HT(2A) receptors was up-regulated in hypertrophic 3T3-L1 adipocytes, which exhibited decreased expression of adiponectin and increased expression of PAI-1. 5-HT(2A) receptor antagonists and suppression of 5-HT(2A) receptor gene expression enhanced adiponectin expression. Activation of Gq negatively regulated adiponectin expression, and inhibition of mitogen-activated protein kinase reversed the Gq-induced effect. Moreover, the 5-HT(2A) receptor blockade reduced PAI-1 expression. These findings indicate that antagonism of 5-HT(2A) receptors in adipocytes could improve the obesity-linked decreases in adiponectin expression and increases in PAI-1 expression.

  5. Plasminogen activator inhibitor-1 4G/5G polymorphism and retinopathy risk in type 2 diabetes: a meta-analysis

    PubMed Central

    2013-01-01

    Background Mounting evidence has suggested that plasminogen activator inhibitor-1 (PAI-1) is a candidate for increased risk of diabetic retinopathy. Studies have reported that insertion/deletion polymorphism in the PAI-1 gene may influence the risk of this disease. To comprehensively address this issue, we performed a meta-analysis to evaluate the association of PAI-1 4G/5G polymorphism with diabetic retinopathy in type 2 diabetes. Methods Data were retrieved in a systematic manner and analyzed using Review Manager and STATA Statistical Software. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of associations. Results Nine studies with 1, 217 cases and 1, 459 controls were included. Allelic and genotypic comparisons between cases and controls were evaluated. Overall analysis suggests a marginal association of the 4G/5G polymorphism with diabetic retinopathy (for 4G versus 5G: OR 1.13, 95%CI 1.01 to 1.26; for 4G/4G versus 5G/5G: OR 1.30, 95%CI 1.04 to 1.64; for 4G/4G versus 5G/5G + 4G/5G: OR 1.26, 95%CI 1.05 to 1.52). In subgroup analysis by ethnicity, we found an association among the Caucasian population (for 4G versus 5G: OR 1.14, 95% CI 1.00 to 1.30; for 4G/4G versus 5G/5G: OR 1.33, 95%CI 1.02 to 1.74; for 4G/4G versus 5G/5G + 4G/5G: OR 1.41, 95%CI 1.13 to 1.77). When stratified by the average duration of diabetes, patients with diabetes histories longer than 10 years have an elevated susceptibility to diabetic retinopathy than those with shorter histories (for 4G/4G versus 5G/5G: OR 1.47, 95%CI 1.08 to 2.00). We also detected a higher risk in hospital-based studies (for 4G/4G versus 5G/5G+4G/5G: OR 1.27, 95%CI 1.02 to 1.57). Conclusions The present meta-analysis suggested that 4G/5G polymorphism in the PAI-1 gene potentially increased the risk of diabetic retinopathy in type 2 diabetes and showed a discrepancy in different ethnicities. A higher susceptibility in patients with longer duration of diabetes (more than 10

  6. Unique secretory dynamics of tissue plasminogen activator and its modulation by plasminogen activator inhibitor-1 in vascular endothelial cells.

    PubMed

    Suzuki, Yuko; Mogami, Hideo; Ihara, Hayato; Urano, Tetsumei

    2009-01-01

    We analyzed the secretory dynamics of tissue plasminogen activator (tPA) in EA.hy926 cells, an established vascular endothelial cell (VEC) line producing GFP-tagged tPA, using total internal reflection-fluorescence (TIR-F) microscopy. tPA-GFP was detected in small granules in EA.hy926 cells, the distribution of which was indistinguishable from intrinsically expressed tPA. Its secretory dynamics were unique, with prolonged (> 5 minutes) retention of the tPA-GFP on the cell surface, appearing as fluorescent spots in two-thirds of the exocytosis events. The rapid disappearance (mostly by 250 ms) of a domain-deletion mutant of tPA-GFP possessing only the signal peptide and catalytic domain indicates that the amino-terminal heavy chain of tPA-GFP is essential for binding to the membrane surface. The addition of PAI-1 dose-dependently facilitated the dissociation of membrane-retained tPA and increased the amounts of tPA-PAI-1 high-molecular-weight complexes in the medium. Accordingly, suppression of PAI-1 synthesis in EA.hy926 cells by siRNA prolonged the dissociation of tPA-GFP, whereas a catalytically inactive mutant of tPA-GFP not forming complexes with PAI-1 remained on the membrane even after PAI-1 treatment. Our results provide new insights into the relationship between exocytosed, membrane-retained tPA and PAI-1, which would modulate cell surface-associated fibrinolytic potential.

  7. Identification of a peroxisome proliferator responsive element (PPRE)-like cis-element in mouse plasminogen activator inhibitor-1 gene promoter

    SciTech Connect

    Chen Jiegen; Li Xi; Huang Haiyan; Liu Honglei; Liu Deguo; Song Tanjing; Ma Chungu; Ma Duan; Song Houyan; Tang Qiqun . E-mail: qqtang@shmu.edu.cn

    2006-09-01

    PAI-1 is expressed and secreted by adipose tissue which may mediate the pathogenesis of obesity-associated cardiovascular complications. Evidence is presented in this report that PAI-1 is not expressed by preadipocyte, but significantly induced during 3T3-L1 adipocyte differentiation and the PAI-1 expression correlates with the induction of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). A peroxisome proliferator responsive element (PPRE)-like cis-element (-206TCCCCCATGCCCT-194) is identified in the mouse PAI-1 gene promoter by electrophoretic mobility shift assay (EMSA) combined with transient transfection experiments; the PPRE-like cis-element forms a specific DNA-protein complex only with adipocyte nuclear extracts, not with preadipocyte nuclear extracts; the DNA-protein complex can be totally competed away by non-labeled consensus PPRE, and can be supershifted with PPAR{gamma} antibody. Mutation of this PPRE-like cis-element can abolish the transactivation of mouse PAI-1 promoter mediated by PPAR{gamma}. Specific PPAR{gamma} ligand Pioglitazone can significantly induce the PAI-1 expression, and stimulate the secretion of PAI-1 into medium.

  8. Plasma levels of plasminogen activator inhibitor type 1, factor VIII, prothrombin activation fragment 1+2, anticardiolipin, and antiprothrombin antibodies are risk factors for thrombosis in hemodialysis patients.

    PubMed

    Molino, Daniela; De Santo, Natale G; Marotta, Rosa; Anastasio, Pietro; Mosavat, Mahrokh; De Lucia, Domenico

    2004-09-01

    Patients with end-stage renal disease are prone to hemorrhagic complications and simultaneously are at risk for a variety of thrombotic complications such as thrombosis of dialysis blood access, the subclavian vein, coronary arteries, cerebral vessel, and retinal veins, as well as priapism. The study was devised for the following purposes: (1) to identify the markers of thrombophilia in hemodialyzed patients, (2) to establish a role for antiphospholipid antibodies in thrombosis of the vascular access, (3) to characterize phospholipid antibodies in hemodialysis patients, and (4) to study the effects of dialysis on coagulation cascade. A group of 20 hemodialysis patients with no thrombotic complications (NTC) and 20 hemodialysis patients with thrombotic complications (TC) were studied along with 400 volunteer blood donors. Patients with systemic lupus erythematosus and those with nephrotic syndrome were excluded. All patients underwent a screening prothrombin time, activated partial thromboplastin time, fibrinogen (Fg), coagulation factors of the intrinsic and extrinsic pathways, antithrombin III (AT-III), protein C (PC), protein S (PS), resistance to activated protein C, prothrombin activation fragment 1+2 (F1+2), plasminogen, tissue type plasminogen activator (t-PA), plasminogen tissue activator inhibitor type-1 (PAI-1), anticardiolipin antibodies type M and G (ACA-IgM and ACA-IgG), lupus anticoagulant antibodies, and antiprothrombin antibodies type M and G (aPT-IgM and aPT-IgG). The study showed that PAI-1, F 1+2, factor VIII, ACA-IgM, and aPT-IgM levels were increased significantly over controls both in TC and NTC, however, they could distinguish patients with thrombotic complications from those without, being increased maximally in the former group. The novelty of the study is represented by the significant aPT increase that was observed in non-systemic lupus erythematosus hemodialysis patients, and particularly in those with thrombotic events. In addition

  9. Fluid Flow Stimulates Tissue Plasminogen Activator Secretion by Cultured Human Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Diamond, S. L.; Eskin, S. G.; McIntire, L. V.

    1989-03-01

    Wall shear stress generated by blood flow may regulate the expression of fibrinolytic proteins by endothelial cells. Tissue plasminogen activator (tPA) and plasminogen activator inhibitor, type 1 (PAI-1) secretion by cultured human endothelial cells were not affected by exposure to venous shear stress (4 dynes/cm2). However, at arterial shear stresses of 15 and 25 dynes/cm2, the tPA secretion rate was 2.1 and 3.0 times greater, respectively, than the basal tPA secretion rate. PAI-1 secretion was unaffected by shear stress over the entire physiological range.

  10. The Effect of PAI-1 4G/5G Polymorphism and Clinical Factors on Coronary Artery Occlusion in Myocardial Infarction

    PubMed Central

    Parpugga, Tajinder Kumar; Tatarunas, Vacis; Skipskis, Vilius; Kupstyte, Nora; Zaliaduonyte-Peksiene, Diana; Lesauskaite, Vaiva

    2015-01-01

    Objective. Data on the impact of PAI-1-675 4G/5G genotype for fibrinolysis during myocardial infarction are inconsistent. The aim of our study was to evaluate the association of clinical and genetic (PAI-1-675 4G/5G polymorphism) factors with coronary artery occlusion in patients with myocardial infarction. Materials and Methods. PAI-1-675 4G/5G detection was achieved by using Sanger sequencing in a sample of patients hospitalized for stent implantation due to myocardial infarction. We categorized the patients into two groups: patients with coronary artery occlusion and patients without coronary artery occlusion according to angiographic evaluation. Results. We identified n = 122 (32.4%) 4G/4G, n = 186 (49.5%) 4G/5G, and n = 68 (18.1%) 5G/5G PAI-1 genotype carriers. Univariate and multivariate analysis showed that only the 4G/5G genotype was associated with coronary artery occlusion (OR: 1.656 and 95% CI: 1.009–2.718, p = 0.046). Conclusions. Our results showed that carriers of PAI-1 4G/5G genotype with myocardial infarction have increased odds of coronary artery occlusion more than 1.6 times in comparison to the carriers of homozygous genotypes. PMID:26273123

  11. Plasminogen Activator System and Breast Cancer: Potential Role in Therapy Decision Making and Precision Medicine

    PubMed Central

    Gouri, Adel; Dekaken, Aoulia; El Bairi, Khalid; Aissaoui, Arifa; Laabed, Nihad; Chefrour, Mohamed; Ciccolini, Joseph; Milano, Gérard; Benharkat, Sadek

    2016-01-01

    Shifting from the historical TNM paradigm to the determination of molecular and genetic subtypes of tumors has been a major improvement to better picture cancerous diseases. The sharper the picture is, the better will be the possibility to develop subsequent strategies, thus achieving higher efficacy and prolonged survival eventually. Recent studies suggest that urokinase-type plasminogen activator (uPA), uPA Receptor (uPAR), and plasmino-gen activator inhibitor-1 (PAI-1) may play a critical role in cancer invasion and metastasis. Consistent with their role in cancer dissemination, high levels of uPA, PAI-1, and uPAR in multiple cancer types correlate with dismal prognosis. In this respect, upfront determination of uPA and PAI-1 as invasion markers has further opened up the possibilities for individualized therapy of breast cancer. Indeed, uPA and PAI-1 could help to classify patients on their risk for metastatic spreading and subsequent relapse, thus helping clinicians in their decision-making process to propose, or not propose, adjuvant therapy. This review covers the implications for cancer diagnosis, prognosis, and therapy of uPA and PAI-1, and therefore how they could be major actors in the development of a precision medicine in breast cancer. PMID:27578963

  12. Plasminogen Activator System and Breast Cancer: Potential Role in Therapy Decision Making and Precision Medicine.

    PubMed

    Gouri, Adel; Dekaken, Aoulia; El Bairi, Khalid; Aissaoui, Arifa; Laabed, Nihad; Chefrour, Mohamed; Ciccolini, Joseph; Milano, Gérard; Benharkat, Sadek

    2016-01-01

    Shifting from the historical TNM paradigm to the determination of molecular and genetic subtypes of tumors has been a major improvement to better picture cancerous diseases. The sharper the picture is, the better will be the possibility to develop subsequent strategies, thus achieving higher efficacy and prolonged survival eventually. Recent studies suggest that urokinase-type plasminogen activator (uPA), uPA Receptor (uPAR), and plasmino-gen activator inhibitor-1 (PAI-1) may play a critical role in cancer invasion and metastasis. Consistent with their role in cancer dissemination, high levels of uPA, PAI-1, and uPAR in multiple cancer types correlate with dismal prognosis. In this respect, upfront determination of uPA and PAI-1 as invasion markers has further opened up the possibilities for individualized therapy of breast cancer. Indeed, uPA and PAI-1 could help to classify patients on their risk for metastatic spreading and subsequent relapse, thus helping clinicians in their decision-making process to propose, or not propose, adjuvant therapy. This review covers the implications for cancer diagnosis, prognosis, and therapy of uPA and PAI-1, and therefore how they could be major actors in the development of a precision medicine in breast cancer. PMID:27578963

  13. Plasminogen activator inhibitor 1: Mechanisms of its synergistic regulation by growth factors

    SciTech Connect

    Song, Xiaoling

    2010-01-01

    My research is on the synergistic regulation of PAI-1 by EGF and TGF-β. The mechanism of synergistic regulation of PAI-1 by EGF and TGF-β are addressed. Methods are described for effective identification of RNA accessible sites for antisense oligodexoxynucleotides (ODNs) and siRNA. In this study effective AS-ODN sequences for both Lcn2 and Bcl2 were identified by in vitro tiled microarray studies. Our results suggest that hybridization of ODN arrays to a target mRNA under physiological conditions might be used as a rapid and reliable in vitro method to accurately identify targets on mRNA molecules for effective antisense and potential siRNA activity in vivo.

  14. A Phytochemical-rich Multivitamin-multimineral Supplement Is Bioavailable and Reduces Serum Oxidized Low-density Lipoprotein, Myeloperoxidase, and Plasminogen Activator Inhibitor-1 in a Four-week Pilot trial of Healthy Individuals

    PubMed Central

    Desai, Anuradha; Lamb, Joseph J.; Chang, Jyh-Lurn; Darland, Gary; Konda, Veera R.

    2014-01-01

    Background: A multivitamin-multimineral supplement combined with a diverse blend of bioactive phytochemicals may provide additional antioxidant capacity and anti-inflammatory property for overall health. This convenient feature may be useful for individuals who want to increase their intake of phytochemicals. Methods: We conducted a pilot study in 15 healthy individuals (8 women and 7 men, mean age 41.7±14.9 years, mean body mass index 28.0±5.6) to investigate the effects of this novel formulation on biomarkers associated with oxidative stress and inflammation. After a 2-week diet that limited intake of fruits and vegetables to 2 servings/day, participants continued with the same restricted diet but began consuming 2 tablets of the study product for the subsequent 4 weeks. Fasting blood samples collected at Week 2 and Week 6 were analyzed and compared using paired t-tests for levels of carotenoids, folate, vitamin B12, homocysteine, oxidized low-density lipoprotein cholesterol (oxLDL), high-sensitivity C-reactive protein (hs-CRP), F2-isoprostane, plasminogen activator inhibitor-1 (PAI-1), and myeloperoxidase. Noninvasive peripheral arterial tonometry (EndoPAT) was also measured. Results: After 4 weeks of supplementation, plasma levels of carotenoids, folate, and vitamin B12, but not homocysteine, were significantly increased (P<.05). Serum levels of oxLDL, PAI-1 and myeloperoxidase were significantly reduced (P<.05), but F2-isoprostane, hs-CRP, and EndoPAT measures were unchanged compared with baseline. The study product was well tolerated. Conclusions: This nutritional supplement is bioavailable as indicated by the significant increase in plasma carotenoids, vitamin B12, and folate levels and may provide health benefits by significantly reducing serum levels of oxLDL, myeloperoxidase, and PAI-1 in healthy individuals. PMID:24808980

  15. Protease Inhibitors from Plants with Antimicrobial Activity

    PubMed Central

    Kim, Jin-Young; Park, Seong-Cheol; Hwang, Indeok; Cheong, Hyeonsook; Nah, Jae-Woon; Hahm, Kyung-Soo; Park, Yoonkyung

    2009-01-01

    Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides). Plants produce a variety of proteins (peptides) that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins). Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides) with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C18 reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents. PMID:19582234

  16. Discovering Anti-platelet Drug Combinations with an Integrated Model of Activator-Inhibitor Relationships, Activator-Activator Synergies and Inhibitor-Inhibitor Synergies

    PubMed Central

    Lombardi, Federica; Golla, Kalyan; Fitzpatrick, Darren J.; Casey, Fergal P.; Moran, Niamh; Shields, Denis C.

    2015-01-01

    Identifying effective therapeutic drug combinations that modulate complex signaling pathways in platelets is central to the advancement of effective anti-thrombotic therapies. However, there is no systems model of the platelet that predicts responses to different inhibitor combinations. We developed an approach which goes beyond current inhibitor-inhibitor combination screening to efficiently consider other signaling aspects that may give insights into the behaviour of the platelet as a system. We investigated combinations of platelet inhibitors and activators. We evaluated three distinct strands of information, namely: activator-inhibitor combination screens (testing a panel of inhibitors against a panel of activators); inhibitor-inhibitor synergy screens; and activator-activator synergy screens. We demonstrated how these analyses may be efficiently performed, both experimentally and computationally, to identify particular combinations of most interest. Robust tests of activator-activator synergy and of inhibitor-inhibitor synergy required combinations to show significant excesses over the double doses of each component. Modeling identified multiple effects of an inhibitor of the P2Y12 ADP receptor, and complementarity between inhibitor-inhibitor synergy effects and activator-inhibitor combination effects. This approach accelerates the mapping of combination effects of compounds to develop combinations that may be therapeutically beneficial. We integrated the three information sources into a unified model that predicted the benefits of a triple drug combination targeting ADP, thromboxane and thrombin signaling. PMID:25875950

  17. Regulatory elements involved in constitutive and phorbol ester-inducible expression of the plasminogen activator inhibitor type 2 gene promoter.

    PubMed Central

    Cousin, E; Medcalf, R L; Bergonzelli, G E; Kruithof, E K

    1991-01-01

    Gene transcription rates and mRNA levels of plasminogen activator inhibitor type 2 (PAI-2) are markedly induced by the tumor promoting agent phorbol 12-myristate 13-acetate (PMA) in human HT1080 fibrosarcoma cells. To identify promoter elements required for basal-, and phorbol ester-inducible expression, deletion mutants of the PAI-1 promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene, were transiently expressed in HT1080 cells. Constitutive CAT activity was expressed from constructs containing more than 215 bp of promoter sequence, whereas deletion to position -91 bp abolished CAT gene expression. Treatment of transfected cells with PMA resulted in a three- to ten-fold increase in CAT expression from all constructs except from the construct shortened to position -91. DNAse1 protection analysis of the promoter region between -215 and the transcription initiation site revealed numerous protected regions, including two AP1-like binding sites (AP1a and AP1b) and one CRE-like element. Site-directed mutagenesis of the AP1a site or of the CRE-like site resulted in the loss of basal CAT activity and abolished the PMA effect, whereas mutagenesis of AP1b only partially inhibited basal and PMA-mediated expression. Our results suggest that the PAI-2 promoter contains at least two elements required for basal gene transcription and PMA-mediated induction. Images PMID:1650454

  18. Plasminogen activator inhibitor-I-related regulation of procollagen I ({alpha}{sub 1} and {alpha}{sub 2}) by antitransforming growth factor-{beta}{sub 1} treatment during radiation-impaired wound healing

    SciTech Connect

    Schultze-Mosgau, Stefan; Thorwarth, Michael; Roedel, Franz; Melnychenko, Ivan; Grabenbauer, Gerhard G.; Amann, Kerstin; Wehrhan, Falk

    2006-01-01

    Purpose: Plasminogen activator inhibitor (PAI)-1 mediates transforming growth factor-{beta}{sub 1} (TGF-{beta}{sub 1})-related signaling by stimulating collagen Type I synthesis in radiation-impaired wound healing. The regulation of {alpha}(I)-procollagen is contradictory in fibroblasts of different fibrotic lesions. It is not known whether anti-TGF-{beta}{sub 1} treatment specifically inhibits {alpha}(I)-procollagen synthesis. We used an experimental wound healing study to address anti-TGF-{beta}{sub 1}-associated influence on {alpha}(I)-procollagen synthesis. Methods and Materials: A free flap was transplanted into the preirradiated (40 Gy) or nonirradiated neck region of Wistar rats: Group 1 (n = 8) surgery alone; Group 2 (n = 14) irradiation and surgery; Group 3 (n = 8) irradiation and surgery and anti-TGF-{beta}{sub 1} treatment. On the 14th postoperative day, skin samples were processed for fibroblast culture, in situ hybridization for TGF-{beta}{sub 1}, immunohistochemistry, and immunoblotting for PAI-1, {alpha}{sub 1}/{alpha}{sub 2}(I)-procollagen. Results: Anti-TGF-{beta}{sub 1} significantly reduced TGF-{beta}{sub 1} mRNA (p < 0.05) and PAI-1 expression (p < 0.05). Anti-TGF-{beta}{sub 1} treatment in vivo significantly reduced {alpha}{sub 1}(I)-procollagen protein (p < 0.05) and the number of expressing cells (p < 0.05) in contrast to significantly increased (p < 0.05) {alpha}{sub 2}(I)-procollagen expression. Conclusion: These results emphasize anti-TGF-{beta}{sub 1} treatment to reduce radiation-induced fibrosis by decreasing {alpha}{sub 1}(I)-procollagen synthesis in vivo. {alpha}{sub 1}(I)-procollagen and {alpha}{sub 2}(I)-procollagen might be differentially regulated by anti-TGF-{beta}{sub 1} treatment. Increased TGF-{beta} signaling in irradiated skin fibroblasts seemed to be reversible, as shown by a reduction in PAI-1 expression after anti-TGF-{beta}{sub 1} treatment.

  19. Effects on coagulation and fibrinolysis induced by influenza in mice with a reduced capacity to generate activated protein C and a deficiency in plasminogen activator inhibitor type 1.

    PubMed

    Keller, Tymen T; van der Sluijs, Koen F; de Kruif, Martijn D; Gerdes, Victor E A; Meijers, Joost C M; Florquin, Sandrine; van der Poll, Tom; van Gorp, Eric C M; Brandjes, Dees P M; Büller, Harry R; Levi, Marcel

    2006-11-24

    Influenza infections increase the risk of diseases associated with a prothrombotic state, such as venous thrombosis and atherothrombotic diseases. However, it is unclear whether influenza leads to a prothrombotic state in vivo. To determine whether influenza activates coagulation, we measured coagulation and fibrinolysis in influenza-infected C57BL/6 mice. We found that influenza increased thrombin generation, fibrin deposition, and fibrinolysis. In addition, we used various anti- and prothrombotic models to study pathways involved in the influenza-induced prothrombotic state. A reduced capacity to generate activated protein C in TM(pro/pro) mice increased thrombin generation and fibrinolysis, whereas treatment with heparin decreased thrombin generation in influenza-infected C57Bl/6 mice. Thrombin generation was not changed in hyperfibrinolytic mice, deficient in plasminogen activator inhibitor type-1 (PAI-1(-/-)); however, increased fibrin degradation was seen. Treatment with tranexamic acid reduced fibrinolysis, but thrombin generation was unchanged. We conclude that influenza infection generates thrombin, increased by reduced levels of protein C and decreased by heparin. The fibrinolytic system appears not to be important for thrombin generation. These findings suggest that influenza leads to a prothrombotic state by coagulation activation. Heparin treatment reduces the influenza induced prothrombotic state. PMID:17068293

  20. Impact of guideline-based use of uPA/PAI-1 on patient outcome in intermediate-risk early breast cancer.

    PubMed

    Kolben, Thomas; Augustin, D; Armbrust, R; Kolben, T M; Degenhardt, T; Burgmann, M; Goess, C; Ditsch, N; Kates, R; Harbeck, N; Wuerstlein, R

    2016-01-01

    The purpose of this study was to evaluate the influence of guideline-based prospective use of uPA/PAI-1 on clinical outcome in an intermediate-risk cohort of breast cancer patients. We analyzed 381 consecutive primary breast cancer patients (2003-2011) at the breast center Ostbayern meeting the following criteria: M0/N0/estrogen receptor (ER)+/G2. Clinical-pathological data, uPA/PAI-1, and follow-up data were collected. Decisions for adjuvant chemotherapy were made upon consideration of prospectively measured uPA/PAI-1. Observed disease-free survival (DFS) and overall survival (OS) were calculated by Kaplan-Meier estimates. Using guideline-based analysis of uPA/PAI-1, treatment with adjuvant chemotherapy was avoided in 86.5 % of patients with low uPA/PAI-1, i.e., 38.8 % of the total patient collective. Median follow-up was 52.5 months. Five-year relapse-free survival in intermediate-risk patients (N0, G2) without chemotherapy was 99 %. Five-year overall survival including all causes of death was 95 %. By using uPA/PAI-1, adjuvant chemotherapy can be avoided in a major part of patients with intermediate-risk breast cancer. Nevertheless, DFS and OS of these patients at 5 years remain excellent. The potential, but hardly measurable, benefit of adjuvant chemotherapy has to be set in contrast with its associated side effects and increased morbidity. Patients with high uPA/PAI-1 show benefit from chemotherapy. In this subgroup, a very good OS was observed as well. These findings strongly support the use of uPA/PAI-1 together with clinic-pathological parameters as an evidence-based, clinically relevant and inexpensive decision tool in the routine of a breast center.

  1. Plasma levels of plasminogen activator inhibitor type 1, factor VIII, prothrombin activation fragment 1+2, anticardiolipin, and antiprothrombin antibodies are risk factors for thrombosis in hemodialysis patients.

    PubMed

    Molino, Daniela; De Santo, Natale G; Marotta, Rosa; Anastasio, Pietro; Mosavat, Mahrokh; De Lucia, Domenico

    2004-09-01

    Patients with end-stage renal disease are prone to hemorrhagic complications and simultaneously are at risk for a variety of thrombotic complications such as thrombosis of dialysis blood access, the subclavian vein, coronary arteries, cerebral vessel, and retinal veins, as well as priapism. The study was devised for the following purposes: (1) to identify the markers of thrombophilia in hemodialyzed patients, (2) to establish a role for antiphospholipid antibodies in thrombosis of the vascular access, (3) to characterize phospholipid antibodies in hemodialysis patients, and (4) to study the effects of dialysis on coagulation cascade. A group of 20 hemodialysis patients with no thrombotic complications (NTC) and 20 hemodialysis patients with thrombotic complications (TC) were studied along with 400 volunteer blood donors. Patients with systemic lupus erythematosus and those with nephrotic syndrome were excluded. All patients underwent a screening prothrombin time, activated partial thromboplastin time, fibrinogen (Fg), coagulation factors of the intrinsic and extrinsic pathways, antithrombin III (AT-III), protein C (PC), protein S (PS), resistance to activated protein C, prothrombin activation fragment 1+2 (F1+2), plasminogen, tissue type plasminogen activator (t-PA), plasminogen tissue activator inhibitor type-1 (PAI-1), anticardiolipin antibodies type M and G (ACA-IgM and ACA-IgG), lupus anticoagulant antibodies, and antiprothrombin antibodies type M and G (aPT-IgM and aPT-IgG). The study showed that PAI-1, F 1+2, factor VIII, ACA-IgM, and aPT-IgM levels were increased significantly over controls both in TC and NTC, however, they could distinguish patients with thrombotic complications from those without, being increased maximally in the former group. The novelty of the study is represented by the significant aPT increase that was observed in non-systemic lupus erythematosus hemodialysis patients, and particularly in those with thrombotic events. In addition

  2. Plasminogen mRNA induction in the mouse brain after kainate excitation: codistribution with plasminogen activator inhibitor-2 (PAI-2) mRNA.

    PubMed

    Sharon, Ronit; Abramovitz, Rene; Miskin, Ruth

    2002-08-15

    Plasminogen (Plg), which can be converted to the active protease plasmin by plasminogen activators, has been previously implicated in brain plasticity and in toxicity inflicted in hippocampal pyramidal neurons by kainate. Here we have localized Plg. mRNA through in situ hybridization in brain cryosections derived from normal adult mice or after kainate injection (i.p.). The results indicated that Plg mRNA was undetectable in the normal brain, but after kainate injection it was induced in neuronal cells in multiple, but specific areas, including layers II-III of the neocortex; the olfactory bulb, anterior olfactory nucleus, and the piriform cortex; the caudate/putamen and accumbens nucleus shell; throughout the amygdaloid complex; and in the CAI/CA3 subfields of the hippocampus. Interestingly, this distribution pattern coincided with what we have recently described for the plasminogen activator inhibitor-2 (PAI-2) mRNA, however differing from that of the plasminogen activator inhibitor-1 (PAI-1) mRNA, as also shown here. These results suggest that enhanced Plg gene expression could be involved in events associated with olfactory, striatal, and limbic structures. Furthermore, because PAI-2 is thought to intracellularly counteract cytotoxic events, our results raise the possibility that PAI-2 can act in the brain as an intracellular neuroprotector against potential plasmin-mediated toxicity.

  3. Increased expression of matrix metalloproteinases (MMP)-2, MMP-9, and the urokinase-type plasminogen activator is associated with progression from benign to advanced ovarian cancer.

    PubMed

    Schmalfeldt, B; Prechtel, D; Härting, K; Späthe, K; Rutke, S; Konik, E; Fridman, R; Berger, U; Schmitt, M; Kuhn, W; Lengyel, E

    2001-08-01

    Proteases are linked to the malignant phenotype of different solid tumors. Therefore, the expression of the matrix metalloproteinase (MMP)-2 and MMP-9 and of the serine protease urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor type 1 (PAI-1) in the progression of ovarian cancer was investigated. Gelatinolytic activity and protein expression of MMP-2 and MMP-9 were analyzed in tissue extracts of 19 cystadenomas and 18 low malignant potential (LMP) tumors, as well as 41 primary tumors of advanced ovarian cancer stage International Federation of Gynecology and Obstetrics IIIc/IV and their corresponding omentum metastases by quantitative gelatin zymography and Western blot. In the same tissue extracts, antigen levels of uPA and its inhibitor PAI-1 were determined by ELISA. Protein expression of pro-MMP-2 (72 kDa) and pro-MMP-9 (92 kDa as well as antigen levels of uPA and PAI-1 were low in benign ovarian tumors but increased significantly from LMP tumors to advanced ovarian cancers. The highest values of all of the proteolytic factors were detected in omentum metastases. Active MMP-2 enzyme (62 kDa) was detected only in ovarian cancer (66%) and corresponding metastases (93%) but never in benign or LMP tumors. The activation rate of MMP-2 to its active isoform was higher in the metastases. Comparing both proteolytic systems, higher PAI-1 concentrations were consistently found in cancers with high pro-MMP-9 expression. These data indicate that members of the plasminogen activator system, as well as the metalloproteinases MMP-2/9, increase with growing malignant potential of ovarian tumors. These findings are of particular relevance to the development of protease inhibitors as new therapeutic approaches in ovarian cancer.

  4. The tissue plasminogen activator-plasminogen proteolytic cascade accelerates amyloid-beta (Abeta) degradation and inhibits Abeta-induced neurodegeneration.

    PubMed

    Melchor, Jerry P; Pawlak, Robert; Strickland, Sidney

    2003-10-01

    Accumulation of the amyloid-beta (Abeta) peptide depends on both its generation and clearance. To better define clearance pathways, we have evaluated the role of the tissue plasminogen activator (tPA)-plasmin system in Abeta degradation in vivo. In two different mouse models of Alzheimer's disease, chronically elevated Abeta peptide in the brain correlates with the upregulation of plasminogen activator inhibitor-1 (PAI-1) and inhibition of the tPA-plasmin system. In addition, Abeta injected into the hippocampus of mice lacking either tPA or plasminogen persists, inducing PAI-1 expression and causing activation of microglial cells and neuronal damage. Conversely, Abeta injected into wild-type mice is rapidly cleared and does not cause neuronal degeneration. Thus, the tPA-plasmin proteolytic cascade aids in the clearance of Abeta, and reduced activity of this system may contribute to the progression of Alzheimer's disease.

  5. Internalization of oligodeoxynucleotide antisense to type-1 plasminogen activator inhibitor mRNA in endothelial cells: a three-dimensional reconstruction by confocal microscopy.

    PubMed

    Wyroba, E; Pawlowska, Z; Kobylanska, A; Pluskota, E; Maszewska, M; Stec, W J; Cierniewski, C S

    1996-01-01

    A three-dimensional reconstruction analysis of localization of phosphodiester and phosphorothioate oligonucleotide antisense to type-1 plasminogen activator inhibitor (PAI-1) mRNA within endothelial cells is described. When EA.hy 926 cells were incubated with fluorescently labelled phosphodiester (PO-16) or phosphorothioate (PS-16) oligonucleotides at low, not cytotoxical concentrations, the relative brightness composition of the images of the particular samples was much higher for PS-16 than PO-16 and dependent upon the extracellular concentration and the incubation time. The 3-D reconstructions based on the series of optical sections of the samples, spaced every 1.5 microns, showed the punctuate accumulation of the oligonucleotides and a striking difference in a spatial distribution between PO-16 and PS-16 within the cytoplasm. Even after 24 h incubation of endothelial cells with 2.5 microM of PO-16 and PS-16 oligonucleotides, there was a predominant oligonucleotide localization within the cytoplasm and only traces of oligonucleotides could be seen in the cell nucleus and/or perinuclear organelles.

  6. Variable Resistance to Plasminogen Activator Initiated Fibrinolysis for Intermediate-Risk Pulmonary Embolism

    PubMed Central

    Stubblefield, William B.; Alves, Nathan J.; Rondina, Matthew T.; Kline, Jeffrey A.

    2016-01-01

    Background We examine the clinical significance and biomarkers of tissue plasminogen activator (tPA)-catalyzed clot lysis time (CLT) in patients with intermediate-risk pulmonary embolism (PE). Methods Platelet-poor, citrated plasma was obtained from patients with PE. Healthy age- and sex-matched patients served as disease-negative controls. Fibrinogen, α2-antiplasmin, plasminogen, thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen activator Inhibitor 1 (PAI-1), thrombin time and D-dimer were quantified. Clotting was induced using CaCl2, tissue factor, and phospholipid. Lysis was induced using 60 ng/mL tPA. Time to 50% clot lysis (CLT) was assessed by both thromboelastography (TEG) and turbidimetry (A405). Results Compared with disease-negative controls, patients with PE exhibited significantly longer mean CLT on TEG (+2,580 seconds, 95% CI 1,380 to 3,720 sec). Patients with PE and a short CLT who were treated with tenecteplase had increased risk of bleeding, whereas those with long CLT had significantly worse exercise tolerance and psychometric testing for quality of life at 3 months. A multivariate stepwise removal regression model selected PAI-1 and TAFI as predictive biomarkers of CLT. Conclusion The CLT from TEG predicted increased risk of bleeding and clinical failure with tenecteplase treatment for intermediate-risk PE. Plasmatic PAI-1 and TAFI were independent predictors of CLT. PMID:26866684

  7. Tissue factor pathway inhibitor prevents airway obstruction, respiratory failure and death due to sulfur mustard analog inhalation

    SciTech Connect

    Rancourt, Raymond C. Veress, Livia A. Ahmad, Aftab Hendry-Hofer, Tara B. Rioux, Jacqueline S. Garlick, Rhonda B. White, Carl W.

    2013-10-01

    Sulfur mustard (SM) inhalation causes airway injury, with enhanced vascular permeability, coagulation, and airway obstruction. The objective of this study was to determine whether recombinant tissue factor pathway inhibitor (TFPI) could inhibit this pathogenic sequence. Methods: Rats were exposed to the SM analog 2-chloroethyl ethyl sulfide (CEES) via nose-only aerosol inhalation. One hour later, TFPI (1.5 mg/kg) in vehicle, or vehicle alone, was instilled into the trachea. Arterial O{sub 2} saturation was monitored using pulse oximetry. Twelve hours after exposure, animals were euthanized and bronchoalveolar lavage fluid (BALF) and plasma were analyzed for prothrombin, thrombin–antithrombin complex (TAT), active plasminogen activator inhibitor-1 (PAI-1) levels, and fluid fibrinolytic capacity. Lung steady-state PAI-1 mRNA was measured by RT-PCR analysis. Airway-capillary leak was estimated by BALF protein and IgM, and by pleural fluid measurement. In additional animals, airway cast formation was assessed by microdissection and immunohistochemical detection of airway fibrin. Results: Airway obstruction in the form of fibrin-containing casts was evident in central conducting airways of rats receiving CEES. TFPI decreased cast formation, and limited severe hypoxemia. Findings of reduced prothrombin consumption, and lower TAT complexes in BALF, demonstrated that TFPI acted to limit thrombin activation in airways. TFPI, however, did not appreciably affect CEES-induced airway protein leak, PAI-1 mRNA induction, or inhibition of the fibrinolytic activity present in airway surface liquid. Conclusions: Intratracheal administration of TFPI limits airway obstruction, improves gas exchange, and prevents mortality in rats with sulfur mustard-analog-induced acute lung injury. - Highlights: • TFPI administration to rats after mustard inhalation reduces airway cast formation. • Inhibition of thrombin activation is the likely mechanism for limiting casts. • Rats given TFPI

  8. Turing pattern formation in fractional activator-inhibitor systems

    NASA Astrophysics Data System (ADS)

    Henry, B. I.; Langlands, T. A. M.; Wearne, S. L.

    2005-08-01

    Activator-inhibitor systems of reaction-diffusion equations have been used to describe pattern formation in numerous applications in biology, chemistry, and physics. The rate of diffusion in these applications is manifest in the single parameter of the diffusion constant, and stationary Turing patterns occur above a critical value of d representing the ratio of the diffusion constants of the inhibitor to the activator. Here we consider activator-inhibitor systems in which the diffusion is anomalous subdiffusion; the diffusion rates are manifest in both a diffusion constant and a diffusion exponent. A consideration of this problem in terms of continuous-time random walks with sources and sinks leads to a reaction-diffusion system with fractional order temporal derivatives operating on the spatial Laplacian. We have carried out an algebraic stability analysis of the homogeneous steady-state solution in fractional activator-inhibitor systems, with Gierer-Meinhardt reaction kinetics and with Brusselator reaction kinetics. For each class of reaction kinetics we identify a Turing instability bifurcation curve in the two-dimensional diffusion parameter space. The critical value of d , for Turing instabilities, decreases monotonically with the anomalous diffusion exponent between unity (standard diffusion) and zero (extreme subdiffusion). We have also carried out numerical simulations of the governing fractional activator-inhibitor equations and we show that the Turing instability precipitates the formation of complex spatiotemporal patterns. If the diffusion of the activator and inhibitor have the same anomalous scaling properties, then the surface profiles of these patterns for values of d slightly above the critical value varies from smooth stationary patterns to increasingly rough and nonstationary patterns as the anomalous diffusion exponent varies from unity towards zero. If the diffusion of the activator is anomalous subdiffusion but the diffusion of the inhibitor

  9. Turing pattern formation in fractional activator-inhibitor systems.

    PubMed

    Henry, B I; Langlands, T A M; Wearne, S L

    2005-08-01

    Activator-inhibitor systems of reaction-diffusion equations have been used to describe pattern formation in numerous applications in biology, chemistry, and physics. The rate of diffusion in these applications is manifest in the single parameter of the diffusion constant, and stationary Turing patterns occur above a critical value of d representing the ratio of the diffusion constants of the inhibitor to the activator. Here we consider activator-inhibitor systems in which the diffusion is anomalous subdiffusion; the diffusion rates are manifest in both a diffusion constant and a diffusion exponent. A consideration of this problem in terms of continuous-time random walks with sources and sinks leads to a reaction-diffusion system with fractional order temporal derivatives operating on the spatial Laplacian. We have carried out an algebraic stability analysis of the homogeneous steady-state solution in fractional activator-inhibitor systems, with Gierer-Meinhardt reaction kinetics and with Brusselator reaction kinetics. For each class of reaction kinetics we identify a Turing instability bifurcation curve in the two-dimensional diffusion parameter space. The critical value of d , for Turing instabilities, decreases monotonically with the anomalous diffusion exponent between unity (standard diffusion) and zero (extreme subdiffusion). We have also carried out numerical simulations of the governing fractional activator-inhibitor equations and we show that the Turing instability precipitates the formation of complex spatiotemporal patterns. If the diffusion of the activator and inhibitor have the same anomalous scaling properties, then the surface profiles of these patterns for values of d slightly above the critical value varies from smooth stationary patterns to increasingly rough and nonstationary patterns as the anomalous diffusion exponent varies from unity towards zero. If the diffusion of the activator is anomalous subdiffusion but the diffusion of the inhibitor

  10. Interferons Induce STAT1-Dependent Expression of Tissue Plasminogen Activator, a Pathogenicity Factor in Puumala Hantavirus Disease.

    PubMed

    Strandin, Tomas; Hepojoki, Jussi; Laine, Outi; Mäkelä, Satu; Klingström, Jonas; Lundkvist, Åke; Julkunen, Ilkka; Mustonen, Jukka; Vaheri, Antti

    2016-05-15

    Hantaviruses are zoonotic viruses that show various degrees of vasculopathy in humans. In this study, we analyzed the regulation of 2 fibrinolytic parameters, tissue plasminogen activator (tPA) and its physiological inhibitor, plasminogen activator inhibitor 1 (PAI-1), in Puumala hantavirus (PUUV)-infected patients and in human microvascular endothelial cells. We detected strong upregulation of tPA in the acute phase of illness and in PUUV-infected macaques and found the tPA level to positively correlate with disease severity. The median levels of PAI-1 during the acute stage did not differ from those during the recovery phase. In concordance, hantaviruses induced tPA but not PAI-1 in microvascular endothelial cells, and the induction was demonstrated to be dependent on type I interferon. Importantly, type I and II interferons directly upregulated tPA through signal transducer and activator of transcription 1 (STAT1), which regulated tPA gene expression via a STAT1-responsive enhancer element. These results suggest that tPA may be a general factor in the immunological response to viruses.

  11. PAI-1 -675 4G/5G Polymorphism in Association with Diabetes and Diabetic Complications Susceptibility: a Meta-Analysis Study

    PubMed Central

    Yang, Fan; Cui, Dai; Shi, Yun; Shen, Chong; Tang, Wei; Yang, Tao

    2013-01-01

    A meta-analysis was performed to assess the association between the PAI-1 -675 4G/5G polymorphism and susceptibility to diabetes mellitus (DM), diabetic nephropathy (DN), diabetic retinopathy (DR) and diabetic coronary artery disease (CAD). A literature-based search was conducted to identify all relevant studies. The fixed or random effect pooled measure was calculated mainly at the allele level to determine heterogeneity bias among studies. Further stratified analyses and sensitivity analyses were also performed. Publication bias was examined by the modified Begg’s and Egger’s test. Twenty published articles with twenty-seven outcomes were included in the meta-analysis: 6 studies with a total of 1,333 cases and 3,011 controls were analyzed for the PAI-1 -675 4G/5G polymorphism with diabetes risk, 7 studies with 1,060 cases and 1,139 controls for DN risk, 10 studies with 1,327 cases and 1,557 controls for DR and 4 studies with 610 cases and 1,042 controls for diabetic CAD risk respectively. Using allelic comparison (4G vs. 5G), the PAI-1 -675 4G/5G polymorphism was observed to have no significant association with diabetes (REM OR 1.07, 95% CI 0.96, 1.20), DN (REM OR 1.10, 95% CI 0.98, 1.25), DR (REM OR 1.09, 95% CI 0.97, 1.22) or diabetic CAD risk (REM OR 1.07, 95% CI 0.81, 1.42), and similar results were obtained in the dominant, recessive and co-dominant models. Our meta-analyses suggest that the PAI-1 -675 4G/5G polymorphism might not be a risk factor for DM, DN, DR or diabetic CAD risk in the populations investigated. This conclusion warrants confirmation by further studies. PMID:24223897

  12. Effect of aldosterone and glycyrrhetinic acid on the protein expression of PAI-1 and p22(phox) in human mononuclear leukocytes.

    PubMed

    Calò, Lorenzo A; Zaghetto, Francesca; Pagnin, Elisa; Davis, Paul A; De Mozzi, Paola; Sartorato, Paola; Martire, Giuseppe; Fiore, Cristina; Armanini, Decio

    2004-04-01

    Aldosterone excess can produce heart and kidney fibrosis, which seem to be related to a direct effect of aldosterone at the level of specific receptors. We report a direct, mineralocorticoid-mediated effect on the protein expression of two markers of oxidative stress after incubation of mononuclear leukocytes with 1 x 10(-8) M aldosterone (p22(phox)/beta-actin = 1.38 +/- 0.05 and PAI-1/beta-actin = 1.80 +/- 0.05). The same effect was also found with 3 x 10(-5) M glycyrrhetinic acid, the principal constituent of licorice root (p22(phox)/beta-actin = 1.37 +/- 0.97 and PAI-1/beta-actin = 1.80 +/- 0.04). The effect of both aldosterone and glycyrrhetinic acid is blocked by incubation with added 1 x 10(-6) M of receptor-antagonist canrenone. Canrenone alone did not show any effect. PAI-1 related protein was also found using 4 x 10(-9) M aldosterone. Incubations with 1 x 10(-9) M for 3 hours as well as 1 x 10(-8) M aldosterone for 5, 10, and 20 minutes were ineffective for both proteins. These data support the previous finding of an involvement of mononuclear leukocytes in the pathogenesis of the oxidative stress induced by hyperaldosteronism. In addition, the results confirm our previous data on a direct effect of glycyrrhetinic acid at the level of mineralocorticoid receptors. PMID:15070972

  13. Cutaneous wound healing through paradoxical MAPK activation by BRAF inhibitors

    PubMed Central

    Escuin-Ordinas, Helena; Li, Shuoran; Xie, Michael W.; Sun, Lu; Hugo, Willy; Huang, Rong Rong; Jiao, Jing; de-Faria, Felipe Meira; Realegeno, Susan; Krystofinski, Paige; Azhdam, Ariel; Komenan, Sara Marie D.; Atefi, Mohammad; Comin-Anduix, Begoña; Pellegrini, Matteo; Cochran, Alistair J.; Modlin, Robert L.; Herschman, Harvey R.; Lo, Roger S.; McBride, William H.; Segura, Tatiana; Ribas, Antoni

    2016-01-01

    BRAF inhibitors are highly effective therapies for the treatment of BRAFV600-mutated melanoma, with the main toxicity being a variety of hyperproliferative skin conditions due to paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway in BRAF wild-type cells. Most of these hyperproliferative skin changes improve when a MEK inhibitor is co-administered, as it blocks paradoxical MAPK activation. Here we show how the BRAF inhibitor vemurafenib accelerates skin wound healing by inducing the proliferation and migration of human keratinocytes through extracellular signal-regulated kinase (ERK) phosphorylation and cell cycle progression. Topical treatment with vemurafenib in two wound-healing mice models accelerates cutaneous wound healing through paradoxical MAPK activation; addition of a mitogen-activated protein kinase kinase (MEK) inhibitor reverses the benefit of vemurafenib-accelerated wound healing. The same dosing regimen of topical BRAF inhibitor does not increase the incidence of cutaneous squamous cell carcinomas in mice. Therefore, topical BRAF inhibitors may have clinical applications in accelerating the healing of skin wounds. PMID:27476449

  14. Cutaneous wound healing through paradoxical MAPK activation by BRAF inhibitors.

    PubMed

    Escuin-Ordinas, Helena; Li, Shuoran; Xie, Michael W; Sun, Lu; Hugo, Willy; Huang, Rong Rong; Jiao, Jing; de-Faria, Felipe Meira; Realegeno, Susan; Krystofinski, Paige; Azhdam, Ariel; Komenan, Sara Marie D; Atefi, Mohammad; Comin-Anduix, Begoña; Pellegrini, Matteo; Cochran, Alistair J; Modlin, Robert L; Herschman, Harvey R; Lo, Roger S; McBride, William H; Segura, Tatiana; Ribas, Antoni

    2016-08-01

    BRAF inhibitors are highly effective therapies for the treatment of BRAF(V600)-mutated melanoma, with the main toxicity being a variety of hyperproliferative skin conditions due to paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway in BRAF wild-type cells. Most of these hyperproliferative skin changes improve when a MEK inhibitor is co-administered, as it blocks paradoxical MAPK activation. Here we show how the BRAF inhibitor vemurafenib accelerates skin wound healing by inducing the proliferation and migration of human keratinocytes through extracellular signal-regulated kinase (ERK) phosphorylation and cell cycle progression. Topical treatment with vemurafenib in two wound-healing mice models accelerates cutaneous wound healing through paradoxical MAPK activation; addition of a mitogen-activated protein kinase kinase (MEK) inhibitor reverses the benefit of vemurafenib-accelerated wound healing. The same dosing regimen of topical BRAF inhibitor does not increase the incidence of cutaneous squamous cell carcinomas in mice. Therefore, topical BRAF inhibitors may have clinical applications in accelerating the healing of skin wounds.

  15. Activation patterns of coagulation and fibrinolysis in baboons following infusion with lethal or sublethal dose of Escherichia coli.

    PubMed

    de Boer, J P; Creasy, A A; Chang, A; Roem, D; Brouwer, M C; Eerenberg, A J; Hack, C E; Taylor, F B

    1993-01-01

    Administration of low doses endotoxin or tumor necrosis factor (TNF) in human experimental models for sepsis results in transient activation of both coagulation and fibrinolysis and subsequent inhibition of the fibrinolytic system by plasminogen activator inhibitor type 1 (PAI-1). We have investigated in a baboon model for sepsis, whether administration of a lethal or sublethal dose of living E. coli could induce similar activation patterns. Levels of thrombin-antithrombin III (TAT) complexes increased significantly to zeniths of 425 and 33 times the baseline values at t+360 in the lethal and sublethal group, respectively. Activation of fibrinolysis, as reflected by plasmin-alpha 2 antiplasmin (PAP) complexes, in the sublethal group was maximal at t+60 and was increasingly inhibited thereafter in spite of a sustained increase of tissue type plasminogen activator (t-PA) levels. In the lethal group PAP complexes increased to a zenith of 38 times the baseline values at t+240. PAI-1 levels increased to 15 times the baseline values at t+360 in the sublethal group, whereas in the lethal group they increased almost linearly to 20 times the baseline values at t+360. Despite high levels of PAI-1, effective inhibition of the fibrinolysis was not established until at T+240 in the lethal group. The difference in activation patterns of both mediator systems in the sublethal and lethal group of baboons indicate that extensive activation of coagulation contributes to the lethal complications in sepsis.

  16. Enhancement of mRNA expression of tissue-type plasminogen activator by L-threo-3,4-dihydroxyphenylserine in association with ocular dominance plasticity.

    PubMed

    Mataga, N; Imamura, K; Shiomitsu, T; Yoshimura, Y; Fukamauchi, K; Watanabe, Y

    1996-11-01

    Tissue-type plasminogen activator (tPA) plays important roles in the regulation of synaptic plasticity in the hippocampus and cerebellum. We found that the expression of tPA mRNA in the visual cortex was increased significantly by the peripheral administration of L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS; 100 mg/kg, i.p.), which we had previously shown to have a promotive effect on ocular dominance (OD) plasticity. When plasminogen activator inhibitor-1 (PAI-1; 100 muM in an osmotic minipump) was infused into the kitten visual cortex, OD plasticity was suppressed; i.e. a significantly large number of binocular cells was recorded in the PAI-1 infused cortex following monocular deprivation. These results, therefore, suggest that the PA system is involved in the promotive effect of L-threo-DOPS in OD plasticity.

  17. Unexpected Activity of a Novel Kunitz-type Inhibitor

    PubMed Central

    Smith, David; Tikhonova, Irina G.; Jewhurst, Heather L.; Drysdale, Orla C.; Dvořák, Jan; Robinson, Mark W.; Cwiklinski, Krystyna; Dalton, John P.

    2016-01-01

    Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. We identified a novel secreted KT inhibitor associated with the gut and parenchymal tissues of the infective juvenile stage of Fasciola hepatica, a helminth parasite of medical and veterinary importance. Unexpectedly, recombinant KT inhibitor (rFhKT1) exhibited no inhibitory activity toward serine proteases but was a potent inhibitor of the major secreted cathepsin L cysteine proteases of F. hepatica, FhCL1 and FhCL2, and of human cathepsins L and K (Ki = 0.4-27 nm). FhKT1 prevented the auto-catalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pulldown experiments from adult parasite culture medium showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2, and FhCL5. Substitution of the unusual P1 Leu15 within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu15/Arg15) had modest adverse effects on the cysteine protease inhibition but conferred potent activity against the serine protease trypsin (Ki = 1.5 nm). Computational docking and sequence analysis provided hypotheses for the exclusive binding of FhKT1 to cysteine proteases, the importance of the Leu15 in anchoring the inhibitor into the S2 active site pocket, and the inhibitor's selectivity toward FhCL1, FhCL2, and human cathepsins L and K. FhKT1 represents a novel evolutionary adaptation of KT protease inhibitors by F. hepatica, with its prime purpose likely in the regulation of the major parasite-secreted proteases and/or cathepsin L-like proteases of its host. PMID:27422822

  18. Determinants of the activity of beta-lactamase inhibitor combinations.

    PubMed

    Livermore, D M

    1993-01-01

    Inhibitor combinations provide one strategy to overcome beta-lactamase-mediated resistance. Their success depends, obviously, on the inhibitor being able to bind and inactivate the beta-lactamase molecules. Clavulanate, sulbactam and tazobactam are irreversible inactivators of many beta-lactamases, forming covalent complexes which resist hydrolysis. 'Suicide' kinetics are seen with some, but not all, enzymes. All three compounds inactivate staphylococcal penicillinase, the chromosomal beta-lactamases of Proteus vulgaris and Bacteroides spp., and the Class IV beta-lactamases present in some klebsiellae. Tazobactam, but not the other compounds, has moderate activity against some Class I (AmpC) chromosomal beta-lactamases, notably that of Morganella morganii, but not that of Enterobacter cloacae. Both clavulanate and tazobactam are strong inhibitors of the widely distributed TEM and SHV plasmid-mediated beta-lactamases; sulbactam is a weaker inhibitor. Other factors, aside from the affinity of the inhibitor for the enzyme, co-determine the success or failure of inhibition. Potentiation is most readily achieved if little enzyme is produced, and if the organism is very permeable to the inhibitor. Thus, resistance to inhibitor combinations is rare in strains of Haemophilus influenzae and Neisseria gonorrhoeae that produce TEM-beta-lactamase, but is commoner in enterobacteria that produce this enzyme, since these are less permeable and sometimes manufacture very large amounts of enzyme. The partner beta-lactam agent is also important. Irrespective of the inhibitor used, piperacillin is easier to protect against TEM beta-lactamases and the M. morganii Class I enzyme than are ampicillin, amoxycillin or ticarcillin. This may relate to the lower affinity of piperacillin for these enzymes, or to its greater affinity for the bacterial penicillin-binding proteins. Finally, pH can affect the degree of inhibition achieved with sulphones for some beta-lactamases, notably TEM-1

  19. Small Molecule Inhibitors Targeting Activator Protein 1 (AP-1)

    PubMed Central

    2015-01-01

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases. PMID:24831826

  20. Novel Mps1 Kinase Inhibitors with Potent Antitumor Activity.

    PubMed

    Wengner, Antje M; Siemeister, Gerhard; Koppitz, Marcus; Schulze, Volker; Kosemund, Dirk; Klar, Ulrich; Stoeckigt, Detlef; Neuhaus, Roland; Lienau, Philip; Bader, Benjamin; Prechtl, Stefan; Raschke, Marian; Frisk, Anna-Lena; von Ahsen, Oliver; Michels, Martin; Kreft, Bertolt; von Nussbaum, Franz; Brands, Michael; Mumberg, Dominik; Ziegelbauer, Karl

    2016-04-01

    Monopolar spindle 1 (Mps1) has been shown to function as the key kinase that activates the spindle assembly checkpoint (SAC) to secure proper distribution of chromosomes to daughter cells. Here, we report the structure and functional characterization of two novel selective Mps1 inhibitors, BAY 1161909 and BAY 1217389, derived from structurally distinct chemical classes. BAY 1161909 and BAY 1217389 inhibited Mps1 kinase activity with IC50 values below 10 nmol/L while showing an excellent selectivity profile. In cellular mechanistic assays, both Mps1 inhibitors abrogated nocodazole-induced SAC activity and induced premature exit from mitosis ("mitotic breakthrough"), resulting in multinuclearity and tumor cell death. Both compounds efficiently inhibited tumor cell proliferation in vitro (IC50 nmol/L range). In vivo, BAY 1161909 and BAY 1217389 achieved moderate efficacy in monotherapy in tumor xenograft studies. However, in line with its unique mode of action, when combined with paclitaxel, low doses of Mps1 inhibitor reduced paclitaxel-induced mitotic arrest by the weakening of SAC activity. As a result, combination therapy strongly improved efficacy over paclitaxel or Mps1 inhibitor monotreatment at the respective MTDs in a broad range of xenograft models, including those showing acquired or intrinsic paclitaxel resistance. Both Mps1 inhibitors showed good tolerability without adding toxicity to paclitaxel monotherapy. These preclinical findings validate the innovative concept of SAC abrogation for cancer therapy and justify clinical proof-of-concept studies evaluating the Mps1 inhibitors BAY 1161909 and BAY 1217389 in combination with antimitotic cancer drugs to enhance their efficacy and potentially overcome resistance. Mol Cancer Ther; 15(4); 583-92. ©2016 AACR.

  1. Arginine mimetic structures in biologically active antagonists and inhibitors.

    PubMed

    Masic, Lucija Peterlin

    2006-01-01

    Peptidomimetics have found wide application as bioavailable, biostable, and potent mimetics of naturally occurring biologically active peptides. L-Arginine is a guanidino group-containing basic amino acid, which is positively charged at neutral pH and is involved in many important physiological and pathophysiological processes. Many enzymes display a preference for the arginine residue that is found in many natural substrates and in synthetic inhibitors of many trypsin-like serine proteases, e.g. thrombin, factor Xa, factor VIIa, trypsin, and in integrin receptor antagonists, used to treat many blood-coagulation disorders. Nitric oxide (NO), which is produced by oxidation of L-arginine in an NADPH- and O(2)-dependent process catalyzed by isoforms of nitric oxide synthase (NOS), exhibits diverse roles in both normal and pathological physiologies and has been postulated to be a contributor to the etiology of various diseases. Development of NOS inhibitors as well as analogs and mimetics of the natural substrate L-arginine, is desirable for potential therapeutic use and for a better understanding of their conformation when bound in the arginine binding site. The guanidino residue of arginine in many substrates, inhibitors, and antagonists forms strong ionic interactions with the carboxylate of an aspartic acid moiety, which provides specificity for the basic amino acid residue in the active side. However, a highly basic guanidino moiety incorporated in enzyme inhibitors or receptor antagonists is often associated with low selectivity and poor bioavailability after peroral application. Thus, significant effort is focused on the design and preparation of arginine mimetics that can confer selective inhibition for specific trypsin-like serine proteases and NOS inhibitors as well as integrin receptor antagonists and possess reduced basicity for enhanced oral bioavailability. This review will describe the survey of arginine mimetics designed to mimic the function of the

  2. Leucocyte expression of genes implicated in the plasminogen activation cascade is modulated by yoghurt peptides.

    PubMed

    Theodorou, Georgios; Politis, Ioannis

    2016-08-01

    The urokinase-plasminogen activator (u-PA), its receptor (u-PAR) and the inhibitors of u-PA (PAI-1 and PAI-2) provide a multi-molecular system in leucocytes that exerts pleiotropic functions influencing the development of inflammatory and immune responses. The objective of the present study was to examine the ability of water soluble extracts (WSE) obtained from traditional Greek yoghurt made from bovine or ovine milk to modulate the expression of u-PA, u-PAR, PAI-1 and PAI-2 in ovine monocytes and neutrophils. WSE were obtained from 8 commercial traditional type Greek yoghurts made from ovine or bovine milk. WSE upregulated the expression of all 4 u-PA related genes in monocytes but the upregulation was much higher in the PAI-1 (10-fold) than in u-PA and u-PAR (3-4 fold) thus, shifting the system towards inhibition. In line with this observation, WSE reduced total and membrane-bound u-PA activity in monocytes. In neutrophils, WSE caused small (50-60%) but significant (P < 0·05) reductions in expression of u-PAR and PAI-2 but had no effect on expression of u-PA, PAI-1 and on total cell-associated and membrane-bound u-PA activity. WSE from yoghurts made from bovine or ovine milk were essentially equally effective in affecting the u-PA system except for the u-PAR gene in ovine neutrophils that was affected (reduced) by the ovine and not the bovine WSE. In conclusion, peptides present in WSE modulated the expression of u-PA related genes but the effect was much more prominent in monocytes than in neutrophils. PMID:27600972

  3. Unraveling the Pivotal Role of Bradykinin in ACE Inhibitor Activity.

    PubMed

    Taddei, Stefano; Bortolotto, L

    2016-10-01

    Historically, the first described effect of an angiotensin converting enzyme (ACE) inhibitor was an increased activity of bradykinin, one of the substrates of ACE. However, in the subsequent years, molecular models describing the mechanism of action of ACE inhibitors in decreasing blood pressure and cardiovascular risk have focused mostly on the renin-angiotensin system. Nonetheless, over the last 20 years, the importance of bradykinin in regulating vasodilation, natriuresis, oxidative stress, fibrinolysis, inflammation, and apoptosis has become clearer. The affinity of ACE appears to be higher for bradykinin than for angiotensin I, thereby suggesting that ACE inhibitors may be more effective inhibitors of bradykinin degradation than of angiotensin II production. Data describing the effect of ACE inhibition on bradykinin signaling support the hypothesis that the most cardioprotective benefits attributed to ACE inhibition may be due to increased bradykinin signaling rather than to decreased angiotensin II signaling, especially when high dosages of ACE inhibitors are considered. In particular, modulation of bradykinin in the endothelium appears to be a major target of ACE inhibition. These new mechanistic concepts may lead to further development of strategies enhancing the bradykinin signaling. PMID:27260014

  4. Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors

    NASA Astrophysics Data System (ADS)

    Ferreira, Rubén; Nilsson, Jesper R.; Solano, Carlos; Andréasson, Joakim; Grøtli, Morten

    2015-05-01

    REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibitor, we consider the herein presented effector to be a significant step forward in the development of tools for kinase signal transduction studies with spatiotemporal control over inhibitor concentration in situ.

  5. Design, Synthesis and Inhibitory Activity of Photoswitchable RET Kinase Inhibitors

    PubMed Central

    Ferreira, Rubén; Nilsson, Jesper R.; Solano, Carlos; Andréasson, Joakim; Grøtli, Morten

    2015-01-01

    REarranged during Transfection (RET) is a transmembrane receptor tyrosine kinase required for normal development and maintenance of neurons of the central and peripheral nervous systems. Deregulation of RET and hyperactivity of the RET kinase is intimately connected to several types of human cancers, most notably thyroid cancers, making it an attractive therapeutic target for small-molecule kinase inhibitors. Novel approaches, allowing external control of the activity of RET, would be key additions to the signal transduction toolbox. In this work, photoswitchable RET kinase inhibitors based on azo-functionalized pyrazolopyrimidines were developed, enabling photonic control of RET activity. The most promising compound displays excellent switching properties and stability with good inhibitory effect towards RET in cell-free as well as live-cell assays and a significant difference in inhibitory activity between its two photoisomeric forms. As the first reported photoswitchable small-molecule kinase inhibitor, we consider the herein presented effector to be a significant step forward in the development of tools for kinase signal transduction studies with spatiotemporal control over inhibitor concentration in situ. PMID:25944708

  6. Capillary electrophoresis investigations of pET3aPAI-1 DNA involving optimized restriction digestion, laser-induced fluorescence detection, and micropreparative separation

    NASA Astrophysics Data System (ADS)

    Sepaniak, Michael J.; Stebbins, Michael; Todd, April; Gibson, Timothy; Peterson, Cynthia; Diack, Moustopha

    1998-05-01

    This work centers around developing methodologies to isolate the PAI-1 coding sequence of the DNA plasmid pET3a-PAI-1. Size Selective Capillary Electrophoresis (SSCE), using entangled polymer filled small i.d. capillaries, is used to develop digestion conditions (time and enzyme concentration) that provide single cuts (at variable positions) of the plasmid using BstYI restriction enzyme. After obtaining optimum partial digest conditions for this enzyme, digestion with Ndel will produce a mixture of fragments that includes the fragment (1354 bp) which contains the intact region of interest. Sensitive detection is achieved via laser induced fluorescence using running buffers containing intercalating dye. Using small i.d. capillary conditions as a starting point, the SSCE system is increased to the micro-preparative scale using various larger i.d. capillaries. The effects of capillary diameter, applied voltage, injection amount, and sample buffer concentration on separation performance are studied. Subsequently, single or limited numbers of injections of the single cut sample using a relatively large i.d. capillary should prove adequate material for digestion with Ndel prior to PCR amplification of the 1354 bp fragment.

  7. Plasminogen activator inhibitor-1 in cigarette smoke exposure and influenza A virus infection-induced lung injury.

    PubMed

    Bhandary, Yashodhar P; Shetty, Shwetha K; Marudamuthu, Amarnath S; Midde, Krishna K; Ji, Hong-Long; Shams, Homoyoun; Subramaniam, Renuka; Fu, Jian; Idell, Steven; Shetty, Sreerama

    2015-01-01

    Parenchymal lung inflammation and airway and alveolar epithelial cell apoptosis are associated with cigarette smoke exposure (CSE), which contributes to chronic obstructive pulmonary disease (COPD). Epidemiological studies indicate that people exposed to chronic cigarette smoke with or without COPD are more susceptible to influenza A virus (IAV) infection. We found increased p53, PAI-1 and apoptosis in AECs, with accumulation of macrophages and neutrophils in the lungs of patients with COPD. In Wild-type (WT) mice with passive CSE (PCSE), p53 and PAI-1 expression and apoptosis were increased in AECs as was lung inflammation, while those lacking p53 or PAI-1 resisted AEC apoptosis and lung inflammation. Further, inhibition of p53-mediated induction of PAI-1 by treatment of WT mice with caveolin-1 scaffolding domain peptide (CSP) reduced PCSE-induced lung inflammation and reversed PCSE-induced suppression of eosinophil-associated RNase1 (EAR1). Competitive inhibition of the p53-PAI-1 mRNA interaction by expressing p53-binding 3'UTR sequences of PAI-1 mRNA likewise suppressed CS-induced PAI-1 and AEC apoptosis and restored EAR1 expression. Consistent with PCSE-induced lung injury, IAV infection increased p53, PAI-1 and apoptosis in AECs in association with pulmonary inflammation. Lung inflammation induced by PCSE was worsened by subsequent exposure to IAV. Mice lacking PAI-1 that were exposed to IAV showed minimal viral burden based on M2 antigen and hemagglutination analyses, whereas transgenic mice that overexpress PAI-1 without PCSE showed increased M2 antigen and inflammation after IAV infection. These observations indicate that increased PAI-1 expression promotes AEC apoptosis and exacerbates lung inflammation induced by IAV following PCSE.

  8. Plasminogen Activator Inhibitor-1 in Cigarette Smoke Exposure and Influenza A Virus Infection-Induced Lung Injury

    PubMed Central

    Bhandary, Yashodhar P.; Shetty, Shwetha K.; Marudamuthu, Amarnath S.; Midde, Krishna K.; Ji, Hong-Long; Shams, Homoyoun; Subramaniam, Renuka; Fu, Jian; Idell, Steven; Shetty, Sreerama

    2015-01-01

    Parenchymal lung inflammation and airway and alveolar epithelial cell apoptosis are associated with cigarette smoke exposure (CSE), which contributes to chronic obstructive pulmonary disease (COPD). Epidemiological studies indicate that people exposed to chronic cigarette smoke with or without COPD are more susceptible to influenza A virus (IAV) infection. We found increased p53, PAI-1 and apoptosis in AECs, with accumulation of macrophages and neutrophils in the lungs of patients with COPD. In Wild-type (WT) mice with passive CSE (PCSE), p53 and PAI-1 expression and apoptosis were increased in AECs as was lung inflammation, while those lacking p53 or PAI-1 resisted AEC apoptosis and lung inflammation. Further, inhibition of p53-mediated induction of PAI-1 by treatment of WT mice with caveolin-1 scaffolding domain peptide (CSP) reduced PCSE-induced lung inflammation and reversed PCSE-induced suppression of eosinophil-associated RNase1 (EAR1). Competitive inhibition of the p53-PAI-1 mRNA interaction by expressing p53-binding 3’UTR sequences of PAI-1 mRNA likewise suppressed CS-induced PAI-1 and AEC apoptosis and restored EAR1 expression. Consistent with PCSE-induced lung injury, IAV infection increased p53, PAI-1 and apoptosis in AECs in association with pulmonary inflammation. Lung inflammation induced by PCSE was worsened by subsequent exposure to IAV. Mice lacking PAI-1 that were exposed to IAV showed minimal viral burden based on M2 antigen and hemagglutination analyses, whereas transgenic mice that overexpress PAI-1 without PCSE showed increased M2 antigen and inflammation after IAV infection. These observations indicate that increased PAI-1 expression promotes AEC apoptosis and exacerbates lung inflammation induced by IAV following PCSE. PMID:25932922

  9. Parathyroid hormone is not an inhibitor of lipoprotein lipase activity.

    PubMed

    Arnadottir, M; Nilsson-Ehle, P

    1994-01-01

    The reduced lipoprotein lipase (LPL) activities in uraemia are reflected by increased serum triglyceride concentrations and reduced HDL cholesterol concentrations. Both hyperparathyroidism and circulating inhibitor(s) of LPL have been associated with the disturbances of lipid metabolism in uraemia. The aim of the present study was to investigate if parathyroid hormone (PTH) had an inhibitory effect on LPL activity. Plasma post-heparin LPL activities, plasma LPL inhibitory activities, serum PTHintact and serum PTHC-terminal concentrations were analysed in 20 patients on haemodialysis and 20 healthy controls. The effects of purified, human PTHintact and a carboxyterminal fragment of PTH (PTH39-84) on LPL activities in post-heparin plasma from healthy individuals and on the enzyme activity of purified, bovine milk LPL, activated with apolipoprotein CII, were studied. Patients had significantly higher plasma LPL inhibitory activities than controls, but there was no correlation between plasma LPL inhibitory activities and serum PTH concentrations. Neither PTHintact nor PTH39-84 had a significant effect on LPL activities in vitro. Thus there was no evidence of a direct inhibition of LPL activity by PTH under the present in-vivo or in-vitro conditions.

  10. Parathyroid hormone is not an inhibitor of lipoprotein lipase activity.

    PubMed

    Arnadottir, M; Nilsson-Ehle, P

    1994-01-01

    The reduced lipoprotein lipase (LPL) activities in uraemia are reflected by increased serum triglyceride concentrations and reduced HDL cholesterol concentrations. Both hyperparathyroidism and circulating inhibitor(s) of LPL have been associated with the disturbances of lipid metabolism in uraemia. The aim of the present study was to investigate if parathyroid hormone (PTH) had an inhibitory effect on LPL activity. Plasma post-heparin LPL activities, plasma LPL inhibitory activities, serum PTHintact and serum PTHC-terminal concentrations were analysed in 20 patients on haemodialysis and 20 healthy controls. The effects of purified, human PTHintact and a carboxyterminal fragment of PTH (PTH39-84) on LPL activities in post-heparin plasma from healthy individuals and on the enzyme activity of purified, bovine milk LPL, activated with apolipoprotein CII, were studied. Patients had significantly higher plasma LPL inhibitory activities than controls, but there was no correlation between plasma LPL inhibitory activities and serum PTH concentrations. Neither PTHintact nor PTH39-84 had a significant effect on LPL activities in vitro. Thus there was no evidence of a direct inhibition of LPL activity by PTH under the present in-vivo or in-vitro conditions. PMID:7870347

  11. Utility of a single mid-trimester measurement of plasminogen activator Type 1 and fibronectin to predict preeclampsia in pregnancy

    PubMed Central

    Ajibola, S. O.; Adeyemo, T. A.; Afolabi, B. B.; Akanmu, A. S.

    2016-01-01

    Background: Preeclampsia (PE) is the second most common cause of maternal death after obstetric hemorrhage in Africa, a resource-limited region. This study was designed to examine the potential usefulness of a single screening plasma plasminogen activator inhibitor-1 (PAI-1) and fibronectin (FN) level for the prediction of PE in pregnant women. Materials and Methods: In a cohort of 180 pregnant women who were normotensive at baseline, venous blood samples were obtained before 20 weeks of gestation for the assay of plasma levels of PAI-1 and FN levels measured by enzyme-linked immunoassay technique. Twenty nonpregnant normotensive women were also evaluated as a control group. Outcomes of gestation were evaluated and correlated with the plasma levels of PAI and FN measured at mid-trimester. Mean plasma values of PAI-1 and FN were also compared between the different outcome groups. Results: Plasma PAI-1 level was significantly higher in the pregnant women (8.68 ± 0.56 ng/ml) than in nonpregnant controls (5.55 ± 0.32 ng/ml) (P = 0.01). However, plasma FN did not show any significant difference in pregnant women (2.60 ± 0.37 μg/ml) and nonpregnant controls (2.60 ± 0.23 μg/ml) (P = 0.9). Mid-trimester mean plasma PAI-1 level measured in women who developed PE (7.08 ± 5.49 ng/ml, n = 12) and gestational hypertension (GH) (9.78 ± 6.2 ng/ml, n = 13) was not significantly different in comparison to normotensive pregnant women (8.78 ± 5.63 ng/ml, n = 153) (P = 0.75). Likewise, the mean FN level in women who developed PE was also not significantly different from nonpreeclamptics; however, the FN level in the pregnant women who developed GH was significantly different from women who remained normotensive throughout pregnancy (P = 0.02). Conclusion: Single mid-trimester assessment of PAI-1 and FN levels in maternal plasma was not found to be useful in predicting PE as an outcome of pregnancy in the study population. PMID:27630384

  12. Activator-inhibitor systems on heterogeneous ecological networks

    NASA Astrophysics Data System (ADS)

    Nicolaides, C.; Cueto-Felgueroso, L.; Juanes, R.

    2012-12-01

    The consideration of activator-inhibitor systems as complex networks has broadened our knowledge of non-equilibrium reaction-diffusion processes in heterogeneous systems. For example, the Turing mechanism represents a classical model for the formation of self-organized spatial structures in non-equilibrium activator-inhibitor systems. The study of Turing patterns in networks with heterogeneous connectivity has revealed that, contrary to other models and systems, the segregation process takes place mainly in vertices of low degree. In this paper, we study the formation of vegetation patterns in semiarid ecosystems from the perspective of a heterogeneous interacting ecological network. The structure of ecological networks yields fundamental insight into the ecosystem self-organization. Using simple rules for the short-range activation and global inhibition, we reconstruct the observed power-law distribution of vegetation patch size that has been observed in semiarid ecosystems like the Kalahari transect.

  13. Antimalarial activity of HIV-1 protease inhibitor in chromone series.

    PubMed

    Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

    2014-12-01

    Increasing parasite resistance to nearly all available antimalarial drugs becomes a serious problem to human health and necessitates the need to continue the search for new effective drugs. Recent studies have shown that clinically utilized HIV-1 protease (HIV-1 PR) inhibitors can inhibit the in vitro and in vivo growth of Plasmodium falciparum. In this study, a series of chromone derivatives possessing HIV-1 PR inhibitory activity has been tested for antimalarial activity against P. falciparum (K1 multi-drug resistant strain). Chromone 15, the potent HIV-1 PR inhibitor (IC50=0.65μM), was found to be the most potent antimalarial compound with IC50=0.95μM while primaquine and tafenoquine showed IC50=2.41 and 1.95μM, respectively. Molecular docking study of chromone compounds against plasmepsin II, an aspartic protease enzyme important in hemoglobin degradation, revealed that chromone 15 exhibited the higher binding affinity (binding energy=-13.24kcal/mol) than the known PM II inhibitors. Thus, HIV-1 PR inhibitor in chromone series has the potential to be a new class of antimalarial agent. PMID:25462990

  14. Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization

    SciTech Connect

    Meng, Zhen; Gan, Ye-Hua

    2015-05-01

    Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation. - Highlights: • COX-2 inhibitor, celecoxib, could enhance radiosensitization. • Radiation induced PTEN inactivation (phosphorylation) and AKT activation. • COX-2 inhibitor induced PTEN expression and activation, and inactivated AKT. • COX-2 inhibitor enhanced radiosensitization through activating PTEN.

  15. Characterization of novel MPS1 inhibitors with preclinical anticancer activity

    PubMed Central

    Jemaà, M; Galluzzi, L; Kepp, O; Senovilla, L; Brands, M; Boemer, U; Koppitz, M; Lienau, P; Prechtl, S; Schulze, V; Siemeister, G; Wengner, A M; Mumberg, D; Ziegelbauer, K; Abrieu, A; Castedo, M; Vitale, I; Kroemer, G

    2013-01-01

    Monopolar spindle 1 (MPS1), a mitotic kinase that is overexpressed in several human cancers, contributes to the alignment of chromosomes to the metaphase plate as well as to the execution of the spindle assembly checkpoint (SAC). Here, we report the identification and functional characterization of three novel inhibitors of MPS1 of two independent structural classes, N-(4-{2-[(2-cyanophenyl)amino][1,2,4]triazolo[1,5-a]pyridin-6-yl}phenyl)-2-phenylacetamide (Mps-BAY1) (a triazolopyridine), N-cyclopropyl-4-{8-[(2-methylpropyl)amino]-6-(quinolin-5-yl)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2a) and N-cyclopropyl-4-{8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2b) (two imidazopyrazines). By selectively inactivating MPS1, these small inhibitors can arrest the proliferation of cancer cells, causing their polyploidization and/or their demise. Cancer cells treated with Mps-BAY1 or Mps-BAY2a manifested multiple signs of mitotic perturbation including inefficient chromosomal congression during metaphase, unscheduled SAC inactivation and severe anaphase defects. Videomicroscopic cell fate profiling of histone 2B-green fluorescent protein-expressing cells revealed the capacity of MPS1 inhibitors to subvert the correct timing of mitosis as they induce a premature anaphase entry in the context of misaligned metaphase plates. Hence, in the presence of MPS1 inhibitors, cells either divided in a bipolar (but often asymmetric) manner or entered one or more rounds of abortive mitoses, generating gross aneuploidy and polyploidy, respectively. In both cases, cells ultimately succumbed to the mitotic catastrophe-induced activation of the mitochondrial pathway of apoptosis. Of note, low doses of MPS1 inhibitors and paclitaxel (a microtubular poison) synergized at increasing the frequency of chromosome misalignments and missegregations in the context of SAC inactivation. This resulted in massive polyploidization followed by the activation of mitotic catastrophe. A

  16. Characterization of novel MPS1 inhibitors with preclinical anticancer activity.

    PubMed

    Jemaà, M; Galluzzi, L; Kepp, O; Senovilla, L; Brands, M; Boemer, U; Koppitz, M; Lienau, P; Prechtl, S; Schulze, V; Siemeister, G; Wengner, A M; Mumberg, D; Ziegelbauer, K; Abrieu, A; Castedo, M; Vitale, I; Kroemer, G

    2013-11-01

    Monopolar spindle 1 (MPS1), a mitotic kinase that is overexpressed in several human cancers, contributes to the alignment of chromosomes to the metaphase plate as well as to the execution of the spindle assembly checkpoint (SAC). Here, we report the identification and functional characterization of three novel inhibitors of MPS1 of two independent structural classes, N-(4-{2-[(2-cyanophenyl)amino][1,2,4]triazolo[1,5-a]pyridin-6-yl}phenyl)-2-phenylacetamide (Mps-BAY1) (a triazolopyridine), N-cyclopropyl-4-{8-[(2-methylpropyl)amino]-6-(quinolin-5-yl)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2a) and N-cyclopropyl-4-{8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2b) (two imidazopyrazines). By selectively inactivating MPS1, these small inhibitors can arrest the proliferation of cancer cells, causing their polyploidization and/or their demise. Cancer cells treated with Mps-BAY1 or Mps-BAY2a manifested multiple signs of mitotic perturbation including inefficient chromosomal congression during metaphase, unscheduled SAC inactivation and severe anaphase defects. Videomicroscopic cell fate profiling of histone 2B-green fluorescent protein-expressing cells revealed the capacity of MPS1 inhibitors to subvert the correct timing of mitosis as they induce a premature anaphase entry in the context of misaligned metaphase plates. Hence, in the presence of MPS1 inhibitors, cells either divided in a bipolar (but often asymmetric) manner or entered one or more rounds of abortive mitoses, generating gross aneuploidy and polyploidy, respectively. In both cases, cells ultimately succumbed to the mitotic catastrophe-induced activation of the mitochondrial pathway of apoptosis. Of note, low doses of MPS1 inhibitors and paclitaxel (a microtubular poison) synergized at increasing the frequency of chromosome misalignments and missegregations in the context of SAC inactivation. This resulted in massive polyploidization followed by the activation of mitotic catastrophe. A

  17. Inhibitors

    MedlinePlus

    ... Community Counts Blood Safety Inhibitors Articles & Key Findings Free Materials Videos Starting the Conversation Playing it Safe A Look at Hemophilia Joint Range of Motion My Story Links to Other Websites ...

  18. High Expression of Urokinase-Type Plasminogen Activator Is Associated with Lymph Node Metastasis of Invasive Ductal Carcinoma of the Breast

    PubMed Central

    Kim, Eun Young; Do, Sung-Im; Hyun, Keehoon; Park, Yong Lai; Kim, Dong-Hoon; Chae, Seoung Wan; Sohn, Jin Hee

    2016-01-01

    Purpose In the present study, we evaluated the levels of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1) by performing immunohistochemical staining to determine whether they were reliable prognostic markers in patients with breast cancer. Methods Demographic and clinicopathological parameters of 214 patients with invasive ductal carcinoma (IDC) and 80 patients with ductal carcinoma in situ (DCIS) who were diagnosed and treated from 2006 to 2010 were analyzed. Tissue microarray was constructed and immunohistochemical staining was performed for each specimen. Results Univariate analyses showed that age at diagnosis, history of hormone replacement therapy, radiation therapy, skin and chest wall invasion, Paget disease, lymphovascular invasion, estrogen receptor positivity, and triple-negative subtype were significantly associated with patient prognosis (p<0.005). Patients with DCIS showed higher PAI-1 expression than patients with IDC (82.5% and 36.2%, respectively; p=0.012). Lymph node metastasis was more frequent in patients with high uPA levels than in patients with low uPA levels (p=0.001). Conclusion Our results suggested that PAI-1 was involved in tumor progression in the early stages of breast cancer, such as DCIS. In addition, our results suggested that high uPA levels were associated with the lymph node metastasis of IDC. PMID:27382391

  19. Characterization of the activation of latent TGF-beta by co-cultures of endothelial cells and pericytes or smooth muscle cells: a self- regulating system

    PubMed Central

    1990-01-01

    The conversion of latent transforming growth factor beta (LTGF-beta) to the active species, transforming growth factor beta (TGF-beta), has been characterized in heterotypic cultures of bovine aortic endothelial (BAE) cells and bovine smooth muscle cells (SMCs). The formation of TGF- beta in co-cultures of BAE cells and SMCs was documented by a specific radioreceptor competition assay, while medium from homotypic cultures of BAE cells or SMCs contained no active TGF-beta as determined by this assay. The concentration of TGF-beta in the conditioned medium of heterotypic co-cultures was estimated to be 400-1,200 pg/ml using the inhibition of BAE cell migration as an assay. Northern blotting of poly A+ RNA extracted from both homotypic and heterotypic cultures of BAE cells and SMCs revealed that BAE cells produced both TGF-beta 1 and TGF- beta 2, while SMCs produced primarily TGF-beta 1. No change in the expression of these two forms of TGF-beta was apparent after 24 h in heterotypic cultures. Time course studies on the appearance of TGF-beta indicated that most of the active TGF-beta was generated within the first 12 h after the establishment of co-cultures. The generation of TGF-beta in co-cultures stimulated the production of the protease inhibitor plasminogen activator inhibitor-1 (PAI-1). The inclusion of neutralizing antibodies to TGF-beta in the co-culture medium blocked the observed increase in PAI-1 levels. The increased expression of PAI- 1 subsequent to TGF-beta formation blocked the activation of the protease required for conversion of LTGF-beta to TGF-beta as the inclusion of neutralizing antibodies to PAI-1 in the co-culture medium resulted in prolonged production of TGF-beta. This effect was lost upon removal of the PAI-1 antibodies. Thus, the activation of LTGF-beta appears to be a self-regulating system. PMID:1696270

  20. Antitcoagulant and antiplatelet activities of scolymoside

    PubMed Central

    Yoon, Eun-Kyung; Ku, Sae-Kwang; Lee, Wonhwa; Kwak, Soyoung; Kang, Hyejin; Jung, Byeongjin; Bae, Jong-Sup

    2015-01-01

    Cyclopia subternata is a medicinal plant commonly used in traditional medicine to relieve pain. Here, the anticoagulant effects of scolymoside, an active compound in C. subternata, were examined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), and the activities of thrombin and activated factor X (FXa). The effects of scolymoside on plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) expression were evaluated in tumor necrosis factor (TNF)-α-activated human endothelial cells. Treatment with scolymoside resulted in prolonged aPTT and PT and the inhibition of thrombin and FXa activities and production. In addition, scolymoside inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. Scolymoside also elicited anticoagulant effects in mice, including a significant reduction in the PAI-1 to t-PA ratio. Collectively, these findings indicate that scolymoside possesses anticoagulant activities and could be developed as a novel anticoagulant. [BMB Reports 2015; 48(10): 577-582] PMID:25887749

  1. Novel cinnoline-based inhibitors of LRRK2 kinase activity.

    PubMed

    Garofalo, Albert W; Adler, Marc; Aubele, Danielle L; Bowers, Simeon; Franzini, Maurizio; Goldbach, Erich; Lorentzen, Colin; Neitz, R Jeffrey; Probst, Gary D; Quinn, Kevin P; Santiago, Pam; Sham, Hing L; Tam, Danny; Truong, Anh P; Ye, Xiaocong M; Ren, Zhao

    2013-01-01

    Leucine rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson's disease (PD). Inhibition of LRRK2 kinase activity is a therapeutic approach that may lead to new treatments for PD. Herein we report the discovery of a series of cinnoline-3-carboxamides that are potent against both wild-type and mutant LRRK2 kinase activity in biochemical assays. These compounds are also shown to be potent inhibitors in a cellular assay and to have good to excellent CNS penetration. PMID:23219325

  2. Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2).

    PubMed

    Anderson, David R; Meyers, Marvin J; Vernier, William F; Mahoney, Matthew W; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Schindler, John F; Reitz, David B; Mourey, Robert J

    2007-05-31

    A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

  3. Thrombophilic genetic factors PAI-1 4G-4G and MTHFR 677TT as risk factors of alcohol, cryptogenic liver cirrhosis and portal vein thrombosis, in a Caucasian population.

    PubMed

    D'Amico, Mario; Pasta, Francesca; Pasta, Linda

    2015-08-15

    The thrombophilic genetic factors (THRGFs), PAI-1 4G-4G, MTHFR 677TT, V Leiden 506Q and Prothrombin 20210A, were studied as risk factors in 865 Caucasian patients with liver cirrhosis, consecutively enrolled from June 2008 to January 2014. A total of 582 HCV, 80 HBV, 94 alcohol, (82 with more than one etiologic factor) and 191 cryptogenic patients with liver cirrhosis had been consecutively enrolled; 243 patients showed portal vein thrombosis (PVT). At least one of the above THRGFs was present in 339/865 patients (39.2%). PAI-1 4G-4G and MTHFR 677TT were the most frequent THRGFs, statistically significant in patients with alcohol, cryptogenic liver cirrhosis, and PVT: respectively 24 and 28, 50 and 73, and 65 and 83 (all chi-square tests>3.84, and p values<0.05). Two logistic regression analysis, using PAI-1 4G-4G and MTHFR 677TT, as dependent variable, confirmed the independent significant relationship of these THRGFs with alcohol, cryptogenic liver cirrhosis and PVT. PAI 1 and MTHFR 677 genotypes, deviated from those expected in populations in Hardy-Weinberg equilibrium (all p values<0.05), in the subgroups of patients with alcohol, cryptogenic liver cirrhosis and presence of PVT. Our study shows the pivotal role of PAI-1 4G-4G and MTHFR 677TT in patients with alcohol, cryptogenic liver cirrhosis, and PVT, in a Caucasian population. In conclusion, thrombo and fibro-genetic mechanisms of PAI-1 4G-4G and MTHFR 677TT, could have a role in the development of liver cirrhosis, mainly in patients without HCV and HBV, and PVT.

  4. Role of the catalytic serine in the interactions of serine proteinases with protein inhibitors of the serpin family. Contribution of a covalent interaction to the binding energy of serpin-proteinase complexes.

    PubMed

    Olson, S T; Bock, P E; Kvassman, J; Shore, J D; Lawrence, D A; Ginsburg, D; Björk, I

    1995-12-15

    The contribution of a covalent bond to the stability of complexes of serine proteinases with inhibitors of the serpin family was evaluated by comparing the affinities of beta-trypsin and the catalytic serine-modified derivative, beta-anhydrotrypsin, for several serpin and non-serpin (Kunitz) inhibitors. Kinetic analyses showed that anhydrotrypsin had little or no ability to compete with trypsin for binding to alpha 1-proteinase inhibitor (alpha 1PI), plasminogen activator inhibitor 1 (PAI-1), antithrombin (AT), or AT-heparin complex when present at up to a 100-fold molar excess over trypsin. By contrast, equimolar levels of anhydrotrypsin blocked trypsin binding to non-serpin inhibitors. Equilibrium binding studies of inhibitor-enzyme interactions monitored by inhibitor displacement of the fluorescence probe, p-aminobenzamidine, from the enzyme active site, confirmed that the binding of serpins to anhydrotrypsin was undetectable in the case of alpha 1PI or AT (KI > 10(-5) M), of low affinity in the case of AT-heparin complex (KI 7-9 x 10(-6) M), and of moderate affinity in the case of PAI-1 (KI 2 x 10(-7) M). This contrasted with the stoichiometric high affinity binding of the serpins to trypsin as well as of the non-serpin inhibitors to both trypsin and anhydrotrypsin. Maximal KI values for serpin-trypsin interactions of 1 to 8 x 10(-11) M, obtained from kinetic analyses of association and dissociation rate constants, indicated that the affinity of serpins for trypsin was minimally 4 to 6 orders of magnitude greater than that of anhydrotrypsin. Anhydrotrypsin, unlike trypsin, failed to induce the characteristic fluorescence changes in a P9 Ser-->Cys PAI-1 variant labeled with a nitrobenzofuran fluorescent probe (NBD) which were shown previously to report the serpin conformational change associated with active enzyme binding. These results demonstrate that a covalent interaction involving the proteinase catalytic serine contributes a major fraction of the binding

  5. Methylene bisphosphonates as the inhibitors of HIV RT phosphorolytic activity.

    PubMed

    Yanvarev, D V; Korovina, A N; Usanov, N N; Khomich, O A; Vepsäläinen, J; Puljula, E; Kukhanova, M K; Kochetkov, S N

    2016-08-01

    The structure-function analysis of 36 methylenebisphosphonates (BPs) as inhibitors of the phosphorolytic activity of native and drug-resistant forms of HIV-1 reverse transcriptase (RT) was performed. It was shown that with the increase of the inhibitory potential of BPs towards the phosphorolytic activity raises their ability to inhibit the RT-catalyzed DNA elongation. Herein, we report the impact of the thymidine analog mutations (TAM) on the activity of bisphosphonates, as well as some structural features of the BPs, allowing them to maintain the inhibitory activity on the enzyme resistant to nucleoside analog therapy. We estimated the Mg(2+)-coordinating group structure, the linker and the aromatic pharmacophore influence on the inhibitory potential of the BPs. Based on the 31 BPs SAR, several BPs with improved inhibitory properties were designed and synthesized. PMID:27230835

  6. Methylene bisphosphonates as the inhibitors of HIV RT phosphorolytic activity.

    PubMed

    Yanvarev, D V; Korovina, A N; Usanov, N N; Khomich, O A; Vepsäläinen, J; Puljula, E; Kukhanova, M K; Kochetkov, S N

    2016-08-01

    The structure-function analysis of 36 methylenebisphosphonates (BPs) as inhibitors of the phosphorolytic activity of native and drug-resistant forms of HIV-1 reverse transcriptase (RT) was performed. It was shown that with the increase of the inhibitory potential of BPs towards the phosphorolytic activity raises their ability to inhibit the RT-catalyzed DNA elongation. Herein, we report the impact of the thymidine analog mutations (TAM) on the activity of bisphosphonates, as well as some structural features of the BPs, allowing them to maintain the inhibitory activity on the enzyme resistant to nucleoside analog therapy. We estimated the Mg(2+)-coordinating group structure, the linker and the aromatic pharmacophore influence on the inhibitory potential of the BPs. Based on the 31 BPs SAR, several BPs with improved inhibitory properties were designed and synthesized.

  7. Factors Associated with Early Platelet Activation in Obese Children

    PubMed Central

    García, Anel Gómez; Núñez, Guillermina García; Sandoval, Martha Eva Viveros; Castellanos, Sergio Gutierrez; Aguilar, Cleto Alvarez

    2014-01-01

    Objective To investigate the factors associated with platelet activation in obese children. Design Cross-sectional study. Setting Department of Pediatrics of Regional Hospital N∘ 1 of Mexican Institute of Social Security in Morelia, Michoacán, Mexico. Participants 79 obese and 64 non-obese children between the ages of 5 and 10 years. Main Outcomes Measures Obese children (body mass index [BMI] >85 in growth curves for Centers for Disease Control/National Center for Health Statistics), and the control group of 64 non-obese children (percentile <85), % body fat, platelet activation was assessed by sP-selectin. Other measures were leptin, uric acid (UA), von Willebrand Factor (vWF), plasminogen activator inhibitor (PAI-1), lipid profile, and glucose. Results Obese children displayed higher plasma sP-selectin, leptin, PAI-1, and vWF than non-obese children. In the univariate logistic regression analysis, leptin, vWF, UA, and high density lipoprotein (HDL), but not with PAI-1, were factors associated with platelet activation. By stepwise linear regression analysis adjusted by sex and age, the best predictor variables for platelet activation were leptin (β:0.381; t:4.665; P=0.0001), vWF (β:0.211; t:2.926; P=0.004), UA (β:0.166; t:2.146; P=0.034), and HDL (β:−0.215; t:−2.819; P=0.006). Conclusions Obese children have a higher risk of developing early platelet activation. Factors associated with platelet activation were Leptin, vWF, UA, and HDL. Further studies involving larger numbers of patients over a longer duration are needed to understand the possible molecular mechanism underlying the association between leptin, vWF, and UA and endothelial activation and/or endothelial damage/dysfunction in obese children and its influence in cardiovascular disease in adults. PMID:24415745

  8. Formation of Tankyrase Inhibitor-Induced Degradasomes Requires Proteasome Activity

    PubMed Central

    Pedersen, Nina Marie; Thorvaldsen, Tor Espen; Schultz, Sebastian Wolfgang; Wenzel, Eva Maria; Stenmark, Harald

    2016-01-01

    In canonical Wnt signaling, the protein levels of the key signaling mediator β-catenin are under tight regulation by the multimeric destruction complex that mediates proteasomal degradation of β-catenin. In colorectal cancer, destruction complex activity is often compromised due to mutations in the multifunctional scaffolding protein Adenomatous Polyposis Coli (APC), leading to a stabilization of β-catenin. Recently, tankyrase inhibitors (TNKSi), a novel class of small molecule inhibitors, were shown to re-establish a functional destruction complex in APC-mutant cancer cell lines by stabilizing AXIN1/2, whose protein levels are usually kept low via poly(ADP-ribosyl)ation by the tankyrase enzymes (TNKS1/2). Surprisingly, we found that for the formation of the morphological correlates of destruction complexes, called degradasomes, functional proteasomes are required. In addition we found that AXIN2 is strongly upregulated after 6 h of TNKS inhibition. The proteasome inhibitor MG132 counteracted TNKSi-induced degradasome formation and AXIN2 stabilization, and this was accompanied by reduced transcription of AXIN2. Mechanistically we could implicate the transcription factor FoxM1 in this process, which was recently shown to be a transcriptional activator of AXIN2. We observed a substantial reduction in TNKSi-induced stabilization of AXIN2 after siRNA-mediated depletion of FoxM1 and found that proteasome inhibition reduced the active (phosphorylated) fraction of FoxM1. This can explain the decreased protein levels of AXIN2 after MG132 treatment. Our findings have implications for the design of in vitro studies on the destruction complex and for clinical applications of TNKSi. PMID:27482906

  9. Proteolytic regulation of epithelial sodium channels by urokinase plasminogen activator: cutting edge and cleavage sites.

    PubMed

    Ji, Hong-Long; Zhao, Runzhen; Komissarov, Andrey A; Chang, Yongchang; Liu, Yongfeng; Matthay, Michael A

    2015-02-27

    Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at ((177)GR↓KR(180)) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically "silent") channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions.

  10. Lumican: a new inhibitor of matrix metalloproteinase-14 activity.

    PubMed

    Pietraszek, Katarzyna; Chatron-Colliet, Aurore; Brézillon, Stéphane; Perreau, Corinne; Jakubiak-Augustyn, Anna; Krotkiewski, Hubert; Maquart, François-Xavier; Wegrowski, Yanusz

    2014-11-28

    We previously showed that lumican regulates MMP-14 expression. The aim of this study was to compare the effect of lumican and decorin on MMP-14 activity. In contrast to decorin, the glycosylated form of lumican was able to significantly decrease MMP-14 activity in B16F1 melanoma cells. Our results suggest that a direct interaction occurs between lumican and MMP-14. Lumican behaves as a competitive inhibitor which leads to a complete blocking of the activity of MMP-14. It binds to the catalytic domain of MMP-14 with moderate affinity (KD∼275 nM). Lumican may protect collagen against MMP-14 proteolysis, thus influencing cell-matrix interaction in tumor progression. PMID:25304424

  11. Chronic monoamine oxidase-B inhibitor treatment blocks monoamine oxidase-A enzyme activity.

    PubMed

    Bartl, Jasmin; Müller, Thomas; Grünblatt, Edna; Gerlach, Manfred; Riederer, Peter

    2014-04-01

    Patients with Parkinson's disease receive selective irreversible monoamine oxidase (MAO)-B inhibitors, but their effects on MAO-A activity are not known during long-term application. We determined MAO-A inhibition in plasma samples from patients with MAO-B inhibitor intake or without MAO-B inhibitor treatment and from healthy controls. We detected a 70 % reduction of MAO-A activity in patients with MAO-B inhibitor therapy in comparison to the other groups. Our results suggest that treatment with MAO-B inhibitor may also influence MAO-A activity in vivo, when administered daily.

  12. Shp2 protein tyrosine phosphatase inhibitor activity of estramustine phosphate and its triterpenoid analogs

    PubMed Central

    Scott, Latanya M.; Chen, Liwei; Daniel, Kenyon G.; Brooks, Wesley H.; Guida, Wayne C.; Lawrence, Harshani R.; Sebti, Said M.; Lawrence, Nicholas J.; Wu, Jie

    2010-01-01

    Shp2 protein tyrosine phosphate (PTP) is a novel target for anticancer drug discovery. We identified estramustine phosphate as a Shp2 PTP inhibitor from the National Cancer Institute Approved Oncology Drug set. A focused structure-activity relationship study indicated that the 17- phosphate group is required for the Shp2 PTP inhibitor activity of estramustine phosphate. A search for estramustine phosphate analogs led to identification of two triperpenoids, enoxolone and celastrol, having Shp2 PTP inhibitor activity. With the previously reported PTP1B inhibitor trodusquemine, our study reveals steroids and triterpenoids with negatively charged phosphate, carboxylate, or sulfonate groups as novel pharmacophores of selective PTP inhibitors. PMID:21193311

  13. Identification of Novel Plasmodium falciparum Hexokinase Inhibitors with Antiparasitic Activity.

    PubMed

    Davis, Mindy I; Patrick, Stephen L; Blanding, Walker M; Dwivedi, Varun; Suryadi, Jimmy; Golden, Jennifer E; Coussens, Nathan P; Lee, Olivia W; Shen, Min; Boxer, Matthew B; Hall, Matthew D; Sharlow, Elizabeth R; Drew, Mark E; Morris, James C

    2016-10-01

    Plasmodium falciparum, the deadliest species of malaria parasites, is dependent on glycolysis for the generation of ATP during the pathogenic red blood cell stage. Hexokinase (HK) catalyzes the first step in glycolysis, transferring the γ-phosphoryl group of ATP to glucose to yield glucose-6-phosphate. Here, we describe the validation of a high-throughput assay for screening small-molecule collections to identify inhibitors of the P. falciparum HK (PfHK). The assay, which employed an ADP-Glo reporter system in a 1,536-well-plate format, was robust with a signal-to-background ratio of 3.4 ± 1.2, a coefficient of variation of 6.8% ± 2.9%, and a Z'-factor of 0.75 ± 0.08. Using this assay, we screened 57,654 molecules from multiple small-molecule collections. Confirmed hits were resolved into four clusters on the basis of structural relatedness. Multiple singleton hits were also identified. The most potent inhibitors had 50% inhibitory concentrations as low as ∼1 μM, and several were found to have low-micromolar 50% effective concentrations against asexual intraerythrocytic-stage P. falciparum parasites. These molecules additionally demonstrated limited toxicity against a panel of mammalian cells. The identification of PfHK inhibitors with antiparasitic activity using this validated screening assay is encouraging, as it justifies additional HTS campaigns with more structurally amenable libraries for the identification of potential leads for future therapeutic development. PMID:27458230

  14. Hypoxia-Ischemia or Excitotoxin-Induced Tissue Plasminogen Activator- Dependent Gelatinase Activation in Mice Neonate Brain Microvessels

    PubMed Central

    Omouendze, Priscilla L.; Henry, Vincent J.; Porte, Baptiste; Dupré, Nicolas; Carmeliet, Peter; Gonzalez, Bruno J.; Marret, Stéphane; Leroux, Philippe

    2013-01-01

    Hypoxia-ischemia (HI) and excitotoxicity are validated causes of neonatal brain injuries and tissue plasminogen activator (t-PA) participates in the processes through proteolytic and receptor-mediated pathways. Brain microvascular endothelial cells from neonates in culture, contain and release more t-PA and gelatinases upon glutamate challenge than adult cells. We have studied t-PA to gelatinase (MMP-2 and MMP-9) activity links in HI and excitotoxicity lesion models in 5 day–old pups in wild type and in t-PA or its inhibitor (PAI-1) genes inactivated mice. Gelatinolytic activities were detected in SDS-PAGE zymograms and by in situ fluorescent DQ-gelatin microscopic zymographies. HI was achieved by unilateral carotid ligature followed by a 40 min hypoxia (8%O2). Excitotoxic lesions were produced by intra parenchymal cortical (i.c.) injections of 10 µg ibotenate (Ibo). Gel zymograms in WT cortex revealed progressive extinction of MMP-2 and MMP-9 activities near day 15 or day 8 respectively. MMP-2 expression was the same in all strains while MMP-9 activity was barely detectable in t-PA−/− and enhanced in PAI-1−/− mice. HI or Ibo produced activation of MMP-2 activities 6 hours post-insult, in cortices of WT mice but not in t-PA−/− mice. In PAI-1−/− mice, HI or vehicle i.c. injection increased MMP-2 and MMP-9 activities. In situ zymograms using DQ-gelatin revealed vessel associated gelatinolytic activity in lesioned areas in PAI-1−/− and in WT mice. In WT brain slices incubated ex vivo, glutamate (200 µM) induced DQ-gelatin activation in vessels. The effect was not detected in t-PA−/−mice, but was restored by concomitant exposure to recombinant t-PA (20 µg/mL). In summary, neonatal brain lesion paradigms and ex vivo excitotoxic glutamate evoked t-PA-dependent gelatinases activation in vessels. Both MMP-2 and MMP-9 activities appeared t-PA-dependent. The data suggest that vascular directed protease inhibition may have neuroprotection

  15. Tissue plasminogen activator involvement in experimental autoimmune myasthenia gravis: aggravation and therapeutic potential.

    PubMed

    Gur-Wahnon, Devorah; Mizrachi, Tehila; Wald-Altman, Shane; Al-Roof Higazi, Abd; Brenner, Talma

    2014-08-01

    Tissue plasminogen activator (tPA), a component of the PA/plasmin system, is elevated in inflammatory areas and plays a role in inflammatory neurological disorders. In the present study we explored the involvement of tPA and the potential immunomodulatory activity of tPA in experimental autoimmune myasthenia gravis (EAMG). Mice deficient in tPA (tPA(-/-)) present with a markedly more severe disease than wild type EAMG mice. In an attempt to treat EAMG with an 18aa peptide derived from the PA system inhibitor (PAI-1), designed to tether out the endogenous inhibitor, a significant suppression of disease severity was demonstrated. The more severe disease in tPA(-/-) mice was accompanied by a higher level of anti-AChR antibodies and increased expression of B-cell markers. In view of the essential role of B-cell activating factor (BAFF) in B-cell maturation, the expression of BAFF family components was tested. An increase in BAFF and BAFF receptor was observed in EAMG tPA(-/-) mice, whereas BCMA expression was reduced, consistent with the increased level of pathogenic antibodies and the more severe disease. Given the importance of T regulatory cells (Tregs) in EAMG, they were evaluated and their number was reduced in tPA(-/-) mice, in which EAMG was aggravated, whereas following PAI-1dp treatment, Tregs were replenished and the disease was ameliorated. The results show the involvement of tPA in EAMG, implying a protective role for tPA in EAMG pathogenesis. The amelioration of EAMG by PAI-1dp treatment suggests that the PA system may be considered a potential site for therapeutic intervention in neuroimmune diseases.

  16. Extravascular fibrin, plasminogen activator, plasminogen activator inhibitors, and airway hyperresponsiveness

    PubMed Central

    Wagers, Scott S.; Norton, Ryan J.; Rinaldi, Lisa M.; Bates, Jason H.T.; Sobel, Burton E.; Irvin, Charles G.

    2004-01-01

    Mechanisms underlying airway hyperresponsiveness are not yet fully elucidated. One of the manifestations of airway inflammation is leakage of diverse plasma proteins into the airway lumen. They include fibrinogen and thrombin. Thrombin cleaves fibrinogen to form fibrin, a major component of thrombi. Fibrin inactivates surfactant. Surfactant on the airway surface maintains airway patency by lowering surface tension. In this study, immunohistochemically detected fibrin was seen along the luminal surface of distal airways in a patient who died of status asthmaticus and in mice with induced allergic airway inflammation. In addition, we observed altered airway fibrinolytic system protein balance consistent with promotion of fibrin deposition in mice with allergic airway inflammation. The airways of mice were exposed to aerosolized fibrinogen, thrombin, or to fibrinogen followed by thrombin. Only fibrinogen followed by thrombin resulted in airway hyperresponsiveness compared with controls. An aerosolized fibrinolytic agent, tissue-type plasminogen activator, significantly diminished airway hyperresponsiveness in mice with allergic airway inflammation. These results are consistent with the hypothesis that leakage of fibrinogen and thrombin and their accumulation on the airway surface can contribute to the pathogenesis of airway hyperresponsiveness. PMID:15232617

  17. Protein inhibitor of activated STAT3 inhibits adipogenic gene expression

    SciTech Connect

    Deng Jianbei; Hua Kunjie; Caveney, Erica J.; Takahashi, Nobuyuki; Harp, Joyce B. . E-mail: jharp@unc.edu

    2006-01-20

    Protein inhibitor of activated STAT3 (PIAS3), a cytokine-induced repressor of signal transducer and activator of transcription 3 (STAT3) and a modulator of a broad array of nuclear proteins, is expressed in white adipose tissue, but its role in adipogenesis is not known. Here, we determined that PIAS3 was constitutively expressed in 3T3-L1 cells at all stages of adipogenesis. However, it translocated from the nucleus to the cytoplasm 4 days after induction of differentiation by isobutylmethylxanthine, dexamethasone, and insulin (MDI). In ob/ob mice, PIAS3 expression was increased in white adipose tissue depots compared to lean mice and was found in the cytoplasm of adipocytes. Overexpression of PIAS3 in differentiating preadipocytes, which localized primarily to the nucleus, inhibited mRNA level gene expression of adipogenic transcription factors C/EBP{alpha} and PPAR{gamma}, as well as their downstream target genes aP2 and adiponectin. PIAS3 also inhibited C/EBP{alpha} promoter activation mediated specifically by insulin, but not dexamethasone or isobutylmethylxanthine. Taken together, these data suggest that PIAS3 may play an inhibitory role in adipogenesis by modulating insulin-activated transcriptional activation events. Increased PIAS3 expression in adipose tissue may play a role in the metabolic disturbances of obesity.

  18. Potentiation of C1 inhibitor by glycosaminoglycans: dextran sulfate species are effective inhibitors of in vitro complement activation in plasma.

    PubMed

    Wuillemin, W A; te Velthuis, H; Lubbers, Y T; de Ruig, C P; Eldering, E; Hack, C E

    1997-08-15

    Activation of the complement system may contribute to the pathogenesis of many diseases. Hence, an effective inhibitor of complement might be useful to reduce tissue damage. Some glycosaminoglycans (GAG), such as heparin, are known to inhibit the interaction of C1q with activators and the assembly of the classical and the alternative pathway C3 convertases. Furthermore, they may potentiate C1 inhibitor-mediated inactivation of C1s. To search for potential complement inhibitors, we systematically investigated the complement inhibitory properties of various synthetic and naturally occurring GAG (dextran sulfates 500,000 and 5,000, heparin, N-acetylheparin, heparan sulfate, dermatan sulfate, and chondroitin sulfates A and C). First, we assessed the effect of GAG on the second-order rate constant of the inactivation of C1s by C1 inhibitor. This rate constant increased 6- to 130-fold in the presence of the GAG, dextran sulfate being the most effective. Second, all tested GAG were found to reduce deposition of C4 and C3 on immobilized aggregated human IgG (AHG) and to reduce fluid phase formation of C4b/c and C3b/c in recalcified plasma upon incubation with AHG. Dextran sulfate again was found to be most effective. We conclude that GAG modulate complement activation in vitro and that the low molecular weight dextran sulfate (m.w. 5000) may be a candidate for pharmacologic manipulation of complement activation via potentiation of C1 inhibitor.

  19. Carbohydrate scaffolds as glycosyltransferase inhibitors with in vivo antibacterial activity.

    PubMed

    Zuegg, Johannes; Muldoon, Craig; Adamson, George; McKeveney, Declan; Le Thanh, Giang; Premraj, Rajaratnam; Becker, Bernd; Cheng, Mu; Elliott, Alysha G; Huang, Johnny X; Butler, Mark S; Bajaj, Megha; Seifert, Joachim; Singh, Latika; Galley, Nicola F; Roper, David I; Lloyd, Adrian J; Dowson, Christopher G; Cheng, Ting-Jen; Cheng, Wei-Chieh; Demon, Dieter; Meyer, Evelyne; Meutermans, Wim; Cooper, Matthew A

    2015-01-01

    The rapid rise of multi-drug-resistant bacteria is a global healthcare crisis, and new antibiotics are urgently required, especially those with modes of action that have low-resistance potential. One promising lead is the liposaccharide antibiotic moenomycin that inhibits bacterial glycosyltransferases, which are essential for peptidoglycan polymerization, while displaying a low rate of resistance. Unfortunately, the lipophilicity of moenomycin leads to unfavourable pharmacokinetic properties that render it unsuitable for systemic administration. In this study, we show that using moenomycin and other glycosyltransferase inhibitors as templates, we were able to synthesize compound libraries based on novel pyranose scaffold chemistry, with moenomycin-like activity, but with improved drug-like properties. The novel compounds exhibit in vitro inhibition comparable to moenomycin, with low toxicity and good efficacy in several in vivo models of infection. This approach based on non-planar carbohydrate scaffolds provides a new opportunity to develop new antibiotics with low propensity for resistance induction. PMID:26194781

  20. Carbohydrate scaffolds as glycosyltransferase inhibitors with in vivo antibacterial activity

    PubMed Central

    Zuegg, Johannes; Muldoon, Craig; Adamson, George; McKeveney, Declan; Le Thanh, Giang; Premraj, Rajaratnam; Becker, Bernd; Cheng, Mu; Elliott, Alysha G.; Huang, Johnny X.; Butler, Mark S.; Bajaj, Megha; Seifert, Joachim; Singh, Latika; Galley, Nicola F.; Roper, David I.; Lloyd, Adrian J.; Dowson, Christopher G.; Cheng, Ting-Jen; Cheng, Wei-Chieh; Demon, Dieter; Meyer, Evelyne; Meutermans, Wim; Cooper, Matthew A.

    2015-01-01

    The rapid rise of multi-drug-resistant bacteria is a global healthcare crisis, and new antibiotics are urgently required, especially those with modes of action that have low-resistance potential. One promising lead is the liposaccharide antibiotic moenomycin that inhibits bacterial glycosyltransferases, which are essential for peptidoglycan polymerization, while displaying a low rate of resistance. Unfortunately, the lipophilicity of moenomycin leads to unfavourable pharmacokinetic properties that render it unsuitable for systemic administration. In this study, we show that using moenomycin and other glycosyltransferase inhibitors as templates, we were able to synthesize compound libraries based on novel pyranose scaffold chemistry, with moenomycin-like activity, but with improved drug-like properties. The novel compounds exhibit in vitro inhibition comparable to moenomycin, with low toxicity and good efficacy in several in vivo models of infection. This approach based on non-planar carbohydrate scaffolds provides a new opportunity to develop new antibiotics with low propensity for resistance induction. PMID:26194781

  1. Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization.

    PubMed

    Meng, Zhen; Gan, Ye-Hua

    2015-05-01

    Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation.

  2. Turing patterns in network-organized activator-inhibitor systems

    NASA Astrophysics Data System (ADS)

    Nakao, Hiroya; Mikhailov, Alexander S.

    2010-07-01

    Turing instability in activator-inhibitor systems provides a paradigm of non-equilibrium self-organization; it has been extensively investigated for biological and chemical processes. Turing instability should also be possible in networks, and general mathematical methods for its treatment have been formulated previously. However, only examples of regular lattices and small networks were explicitly considered. Here we study Turing patterns in large random networks, which reveal striking differences from the classical behaviour. The initial linear instability leads to spontaneous differentiation of the network nodes into activator-rich and activator-poor groups. The emerging Turing patterns become furthermore strongly reshaped at the subsequent nonlinear stage. Multiple coexisting stationary states and hysteresis effects are observed. This peculiar behaviour can be understood in the framework of a mean-field theory. Our results offer a new perspective on self-organization phenomena in systems organized as complex networks. Potential applications include ecological metapopulations, synthetic ecosystems, cellular networks of early biological morphogenesis, and networks of coupled chemical nanoreactors.

  3. Antihormonal activities of 5 alpha-reductase and aromatase inhibitors.

    PubMed

    Zoppi, S; Cocconi, M; Lechuga, M J; Messi, E; Zanisi, M; Motta, M

    1988-10-01

    The problem of developing androgen antagonists has been tackled so far only by synthesizing steroids able to displace testosterone and other androgens from their specific receptor sites. The observation that testosterone has to be converted intracellularly either to 5 alpha-reduced metabolites (DHT, 3 alpha-diol, etc.) or to estrogens, in order to become fully active on androgen-dependent structures (both central and peripheral), has opened the possibility of creating molecules which prevent these conversions, and which could then block the actions of testosterone. The availability of these new compounds has allowed a better understanding of the selective physiological role of each of the metabolites of testosterone, and to provide the basis for the development of new hormone antagonists to be used in those clinical conditions for which an inhibition of the actions of testosterone is foreseen. The usefulness of these enzyme inhibitors is underlined by some examples described in this paper. The results obtained may permit the formulation of the following conclusions: (1) The conversion of testosterone to its 5 alpha-reduced metabolites occurring in the neuroendocrine structures may represent an essential step for the appearance of the inhibitory feedback effect testosterone exerts on LH secretion; (2) Testosterone exhibits its negative feedback effect on FSH secretion as such and not following the local aromatization to estrogens; (3) Testosterone exerts its effect on the intrahypothalamic stores of LHRH acting as such and not following its local conversion either to 5 alpha-reduced metabolites or to estrogenic molecules; (4) Some of the new enzyme inhibitors (e.g. 4-OH-A) may represent an interesting tool for the treatment and/or the prevention of BPH and possibly of other androgen-dependent diseases (prostate carcinoma, acne etc.), as shown by their ability to prevent the in vitro conversion of testosterone to its 5 alpha-reduced metabolites both in the normal

  4. Nonpeptidic HIV protease inhibitors possessing excellent antiviral activities and therapeutic indices. PD 178390: a lead HIV protease inhibitor.

    PubMed

    Prasad, J V; Boyer, F E; Domagala, J M; Ellsworth, E L; Gajda, C; Hamilton, H W; Hagen, S E; Markoski, L J; Steinbaugh, B A; Tait, B D; Humblet, C; Lunney, E A; Pavlovsky, A; Rubin, J R; Ferguson, D; Graham, N; Holler, T; Hupe, D; Nouhan, C; Tummino, P J; Urumov, A; Zeikus, E; Zeikus, G; Gracheck, S J; Erickson, J W

    1999-12-01

    With the insight generated by the availability of X-ray crystal structures of various 5,6-dihydropyran-2-ones bound to HIV PR, inhibitors possessing various alkyl groups at the 6-position of 5,6-dihydropyran-2-one ring were synthesized. The inhibitors possessing a 6-alkyl group exhibited superior antiviral activities when compared to 6-phenyl analogues. Antiviral efficacies were further improved upon introduction of a polar group (hydroxyl or amino) on the 4-position of the phenethyl moiety as well as the polar group (hydroxymethyl) on the 3-(tert-butyl-5-methyl-phenylthio) moiety. The polar substitution is also advantageous for decreasing toxicity, providing inhibitors with higher therapeutic indices. The best inhibitor among this series, (S)-6-[2-(4-aminophenyl)-ethyl]-(3-(2-tert-butyl-5-methyl-phenylsulfa nyl)-4-hydroxy-6-isopropyl-5,6-dihydro-pyran-2-one (34S), exhibited an EC50 of 200 nM with a therapeutic index of > 1000. More importantly, these non-peptidic inhibitors, 16S and 34S, appear to offer little cross-resistance to the currently marketed peptidomimetic PR inhibitors. The selected inhibitors tested in vitro against mutant HIV PR showed a very small increase in binding affinities relative to wild-type HIV PR. Cmax and absolute bioavailability of 34S were higher and half-life and time above EC95 were longer compared to 16S. Thus 34S, also known as PD 178390, which displays good antiviral efficacy, promising pharmacokinetic characteristics and favorable activity against mutant enzymes and CYP3A4, has been chosen for further preclinical evaluation.

  5. MAPK pathway activation leads to Bim loss and histone deacetylase inhibitor resistance: rationale to combine romidepsin with an MEK inhibitor.

    PubMed

    Chakraborty, Arup R; Robey, Robert W; Luchenko, Victoria L; Zhan, Zhirong; Piekarz, Richard L; Gillet, Jean-Pierre; Kossenkov, Andrew V; Wilkerson, Julia; Showe, Louise C; Gottesman, Michael M; Collie, Nathan L; Bates, Susan E

    2013-05-16

    To identify molecular determinants of histone deacetylase inhibitor (HDI) resistance, we selected HuT78 cutaneous T-cell lymphoma (CTCL) cells with romidepsin in the presence of P-glycoprotein inhibitors to prevent transporter upregulation. Resistant sublines were 250- to 385-fold resistant to romidepsin and were resistant to apoptosis induced by apicidin, entinostat, panobinostat, belinostat, and vorinostat. A custom TaqMan array identified increased insulin receptor (INSR) gene expression; immunoblot analysis confirmed increased protein expression and a four- to eightfold increase in mitogen-activated protein kinase (MAPK) kinase (MEK) phosphorylation in resistant cells compared with parental cells. Resistant cells were exquisitely sensitive to MEK inhibitors, and apoptosis correlated with restoration of proapoptotic Bim. Romidepsin combined with MEK inhibitors yielded greater apoptosis in cells expressing mutant KRAS compared with romidepsin treatment alone. Gene expression analysis of samples obtained from patients with CTCL enrolled on the NCI1312 phase 2 study of romidepsin in T-cell lymphoma suggested perturbation of the MAPK pathway by romidepsin. Immunohistochemical analysis of Bim expression demonstrated decreased expression in some skin biopsies at disease progression. These findings implicate increased activation of MEK and decreased Bim expression as a resistance mechanism to HDIs, supporting combination of romidepsin with MEK inhibitors in clinical trials.

  6. A serpin-induced extensive proteolytic susceptibility of urokinase-type plasminogen activator implicates distortion of the proteinase substrate-binding pocket and oxyanion hole in the serpin inhibitory mechanism.

    PubMed

    Egelund, R; Petersen, T E; Andreasen, P A

    2001-02-01

    The formation of stable complexes between serpins and their target serine proteinases indicates formation of an ester bond between the proteinase active-site serine and the serpin P1 residue [Egelund, R., Rodenburg, K.W., Andreasen, P.A., Rasmussen, M.S., Guldberg, R.E. & Petersen, T.E. (1998) Biochemistry 37, 6375-6379]. An important question concerning serpin inhibition is the contrast between the stability of the ester bond in the complex and the rapid hydrolysis of the acyl-enzyme intermediate in general serine proteinase-catalysed peptide bond hydrolysis. To answer this question, we used limited proteolysis to detect conformational differences between free urokinase-type plasminogen activator (uPA) and uPA in complex with plasminogen activator inhibitor-1 (PAI-1). Whereas the catalytic domain of free uPA, pro-uPA, uPA in complex with non-serpin inhibitors and anhydro-uPA in a non-covalent complex with PAI-1 was resistant to proteolysis, the catalytic domain of PAI-1-complexed uPA was susceptible to proteolysis. The cleavage sites for four different proteinases were localized in specific areas of the C-terminal beta-barrel of the catalytic domain of uPA, providing evidence that the serpin inhibitory mechanism involves a serpin-induced massive rearrangement of the proteinase active site, including the specificity pocket, the oxyanion hole, and main-chain binding area, rendering the proteinase unable to complete the normal hydrolysis of the acyl-enzyme intermediate. The distorted region includes the so-called activation domain, also known to change conformation on zymogen activation.

  7. Structure-activity relationship of cyanine tau aggregation inhibitors

    PubMed Central

    Chang, Edward; Congdon, Erin E.; Honson, Nicolette S.; Duff, Karen E.; Kuret, Jeff

    2009-01-01

    A structure-activity relationship for symmetrical cyanine inhibitors of human tau aggregation was elaborated using a filter trap assay. Antagonist activity depended on cyanine heterocycle, polymethine bridge length, and the nature of meso- and N-substituents. One potent member of the series, 3,3’-diethyl-9-methylthiacarbocyanine iodide (compound 11), retained submicromolar potency and had calculated physical properties consistent with blood-brain barrier and cell membrane penetration. Exposure of organotypic slices prepared from JNPL3 transgenic mice (which express human tau harboring the aggregation prone P301L tauopathy mutation) to compound 11 for one week revealed a biphasic dose response relationship. Low nanomolar concentrations decreased insoluble tau aggregates to half those observed in slices treated with vehicle alone. In contrast, high concentrations (≥300 nM) augmented tau aggregation and produced abnormalities in tissue tubulin levels. These data suggest that certain symmetrical carbocyanine dyes can modulate tau aggregation in the slice biological model at concentrations well below those associated with toxicity. PMID:19432420

  8. Direct inhibitors of InhA active against Mycobacterium tuberculosis

    PubMed Central

    Manjunatha, Ujjini H.; Rao, Srinivasa P. S.; Kondreddi, Ravinder Reddy; Noble, Christian G.; Camacho, Luis R.; Tan, Bee H.; Ng, Seow H.; Ng, Pearly Shuyi; Ma, N. L.; Lakshminarayana, Suresh B.; Herve, Maxime; Barnes, S. Whitney; Yu, Weixuan; Kuhen, Kelli; Blasco, Francesca; Beer, David; Walker, John R.; Tonge, Peter J.; Glynne, Richard; Smith, Paul W.; Diagana, Thierry T.

    2015-01-01

    New chemotherapeutic agents are urgently required to combat the global spread of multi-drug resistant tuberculosis (MDR-TB). The mycobacterial enoyl reductase, InhA, is one of the few clinically-validated targets in tuberculosis drug discovery. Here, we report the identification of a new class of direct InhA inhibitors, the 4-hydroxy-2-pyridones, using phenotypic high-throughput whole-cell screening. This class of orally-active compounds showed potent bactericidal activity against common isoniazid-resistant TB clinical isolates. Biophysical studies revealed that 4-hydroxy-2-pyridones bound specifically to InhA in an NADH-dependent manner and blocked the enoyl-substrate binding pocket. The lead compound NITD-916 directly blocked InhA in a dose-dependent manner and showed in vivo efficacy in acute and established mouse models of infection by Mycobacterium tuberculosis. Collectively, our structural and biochemical data open up new avenues for rational structure-guided optimization of the 4-hydroxy-2-pyridone class of compounds for the treatment of MDR-TB. PMID:25568071

  9. Relationships between plasma insulin triglyceride, body mass index, and plasminogen activator inhibitor 1.

    PubMed

    Juhan-Vague, I; Vague, P; Alessi, M C; Badier, C; Valadier, J; Aillaud, M F; Atlan, C

    1987-07-01

    Low fibrinolytic activity, as measured by euglobulin (EFA), has been observed in obese subjects, and hypofibrinolysis may play a role in the pathogenesis of atherosclerosis and its complications. Blood fibrinolytic activity is regulated through a complex system of activators and inhibitors, especially plasminogen activator inhibitors (PA Inhibitors). In a group of 35 non-diabetic subjects with a wide range of body mass index (BMI), EFA was negatively correlated, and PA Inhibitor activity positively correlated, with BMI and plasma insulin levels. In a population of 49 non-diabetic obese women (differing from a control group of normal weight by lower EFA and higher level, of PA Inhibitor activity, plasma insulin and triglyceride), the PA Inhibitor activity was positively correlated with BMI, insulin and triglyceride. The increase in PA Inhibitor activity was associated with a high value of PA Inhibitor 1 antigen measured by an immuno-radiometric assay, indicating that the increased activity was due to a high level of circulating PA Inhibitor 1. Plasma insulin was lowered in obese non-diabetic subjects, without modification of the body weight, by a 24 hour fast or by treatment with Metformin. After 24 hours' fast, ten obese subjects had lower levels of insulin and PA Inhibitor activity and an increase in EFA. Treatment for 15 days by 1.75 g Metformin (or placebo), on a weight maintaining diet, induced, in the Metformin group, a decrease in plasma insulin, triglyceride and PA Inhibitor activity and an increase in EFA, while no change was observed in the placebo group.(ABSTRACT TRUNCATED AT 250 WORDS)

  10. Decrease of fibrinolytic activity in human endothelial cells by arsenite.

    PubMed

    Jiang, Shinn-Jong; Lin, Tsun-Mei; Wu, Hua-Lin; Han, Huai-Song; Shi, Guey-Yueh

    2002-01-01

    Blackfoot disease (BFD) is an endemic peripheral vascular occlusive disease that occurred in the southwest coast of Taiwan. It is believed that arsenic in the drinking water from artesian wells plays an important role in the development of the disease. We have previously shown that BFD patients had significant lower tissue-type plasminogen activator (t-PA) antigen level and higher plasminogen activator inhibitor, Type 1 (PAI-1) antigen level than normal controls. The purpose of this study was to investigate the effects of arsenite on the fibrinolytic and anticoagulant activities of cultured macrovascular and microvascular endothelial cells. Incubation of human microvascular endothelial cells (HMEC-1), but not human umbilical vein endothelial cells (HUVECs), with arsenite caused a decrease of t-PA mRNA level, a rise of both PAI-1 mRNA level and PAI activity. Arsenite could also inhibit the thrombomodulin (TM) mRNA expression and reduce the TM antigen level in HMEC-1. In conclusion, arsenite had a greater effect on HMEC-1 as compared to HUVECs in lowering the fibrinolytic activity and may be responsible for the reduced capacity of fibrinolysis associated with BFD.

  11. Design and synthesis of conformationally restricted inhibitors of active thrombin activatable fibrinolysis inhibitor (TAFIa).

    PubMed

    Brink, Mikael; Dahlén, Anders; Olsson, Thomas; Polla, Magnus; Svensson, Tor

    2014-04-01

    A series of 4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid and 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid derivatives designed as inhibitors of TAFIa has been prepared via a common hydrogenation-alkylation sequence starting from the appropriate benzimidazole and imidazopyridine system. We present a successful design strategy using a conformational restriction approach resulting in potent and selective inhibitors of TAFIa. The X-ray structure of compound 5 in complex with a H333Y/H335Q double mutant TAFI indicate that the conformational restriction is responsible for the observed potency increase. PMID:24588961

  12. Control of DNA replication in a transformed lymphoid cell line: coexistence of activator and inhibitor activities.

    PubMed

    Coffman, F D; Fresa, K L; Oglesby, I; Cohen, S

    1991-12-01

    Proliferating lymphocytes contain an intracellular factor, ADR (activator of DNA replication), which can initiate DNA synthesis in isolated quiescent nuclei. Resting lymphocytes lack ADR activity and contain an intracellular inhibitory factor that suppresses DNA synthesis in normal but not transformed nuclei. In this study we describe a MOLT-4 subline that produces both the activator and inhibitory activities which can be separated by ammonium sulfate fractionation. The inhibitor is heat stable and inhibits ADR-mediated DNA replication in a dose-dependent manner. It does not inhibit DNA polymerase alpha activity. The inhibitor must be present at the initiation of DNA replication to be effective, as it loses most of its effectiveness if it is added after replication has begun. The presence of inhibitory activity in proliferating MOLT-4 cells, taken with the previous observation that inhibitor derived from normal resting cells does not affect DNA synthesis by MOLT-4 nuclei, suggests that failure of a down-regulating signal may play an important role in proliferative disorder. PMID:1934078

  13. Angiotensin-Converting Enzyme Inhibitors and Active Tuberculosis

    PubMed Central

    Wu, Jiunn-Yih; Lee, Meng-Tse Gabriel; Lee, Si-Huei; Lee, Shih-Hao; Tsai, Yi-Wen; Hsu, Shou-Chien; Chang, Shy-Shin; Lee, Chien-Chang

    2016-01-01

    Abstract Numerous epidemiological data suggest that the use of angiotensin-converting enzyme inhibitors (ACEis) can improve the clinical outcomes of pneumonia. Tuberculosis (TB) is an airborne bacteria like pneumonia, and we aimed to find out whether the use of ACEis can decrease the risk of active TB. We conducted a nested case–control analysis by using a 1 million longitudinally followed cohort, from Taiwan national health insurance research database. The rate ratios (RRs) for TB were estimated by conditional logistic regression, and adjusted using a TB-specific disease risk score (DRS) with 71 TB-related covariates. From January, 1997 to December, 2011, a total of 75,536 users of ACEis, and 7720 cases of new active TB were identified. Current use (DRS adjusted RR, 0.87 [95% CI, 0.78–0.97]), but not recent and past use of ACEis, was associated with a decrease in risk of active TB. Interestingly, it was found that chronic use (>90 days) of ACEis was associated with a further decrease in the risk of TB (aRR, 0.74, [95% CI, 0.66–0.83]). There was also a duration response effect, correlating decrease in TB risk with longer duration of ACEis use. The decrease in TB risk was also consistent across all patient subgroups (age, sex, heart failure, cerebrovascular diseases, myocardial infraction, renal diseases, and diabetes) and patients receiving other cardiovascular medicine. In this large population-based study, we found that subjects with recent and chronic use of ACEis were associated with decrease in TB risk. PMID:27175655

  14. The effect of ageing on platelet function and fibrinolytic activity.

    PubMed

    Gleerup, G; Winther, K

    1995-08-01

    Twelve healthy male volunteers, mean age twenty-five, range twenty-one to thirty years, and 12 healthy middle-aged male volunteers mean age fifty-eight, range forty-four to seventy-two years, were tested regarding platelet aggregation induced by adenosine diphosphate and fibrinolytic activity, estimated as euglobulin clot lysis time (ECLT), tissue plasminogen activator (t-PA), and the fast-acting inhibitor against t-PA normally referred to as (PAI-1). Platelet aggregation increased significantly in the middle-aged group as compared with the young, as shown by a decrease in ADP thresholds for irreversible aggregation (P < 0.01). In healthy young volunteers, vigorous cycling exercise by itself caused platelet aggregability to decrease (P < 0.05). Such changes were not observed in the elderly. Fibrinolytic activity decreased significantly in the middle-aged group as shown by a prolongation of the ECLT (P < 0.01) and PAI-1, although not significantly, increased by approximately 100%, whereas t-PA significantly increased in the middle-aged group (P < 0.01). The present results suggest that increasing age is associated with not only increased platelet aggregability but also decreased fibrinolytic activity.

  15. Effect of wine inhibitors on free pineapple stem bromelain activity in a model wine system.

    PubMed

    Esti, Marco; Benucci, Ilaria; Liburdi, Katia; Garzillo, Anna Maria Vittoria

    2011-04-13

    The influence of potential inhibitors, naturally present in wine, on the activity of stem bromelain was investigated in order to evaluate the applicability of this enzyme for protein stabilization in white wine. Bromelain proteolytic activity was tested against a synthetic substrate (Bz-Phe-Val-Arg-pNA) in a model wine system after adding ethanol, sulfur dioxide (SO(2)), skin, seed, and gallic and ellagic tannins at the average range of their concentration in wine. All the inhibitors of stem bromelain activity tested turned out to be reversible. Ethanol was a competitive inhibitor with a rather limited effect. Gallic and ellagic tannins have no inhibitory effect on stem bromelain activity, while both seed and skin tannins were uncompetitive inhibitors. The strongest inhibition effect was revealed for sulfur dioxide, which was a mixed-type inhibitor for the enzyme activity. This study provides useful information relative to a future biotechnological application of stem bromelain in winemaking.

  16. Synergistic Activity of Combined NS5A Inhibitors

    PubMed Central

    Nower, Peter T.; Gao, Min; Fridell, Robert; Wang, Chunfu; Hewawasam, Piyasena; Lopez, Omar; Tu, Yong; Meanwell, Nicholas A.; Belema, Makonen; Roberts, Susan B.; Cockett, Mark; Sun, Jin-Hua

    2015-01-01

    Daclatasvir (DCV) is a first-in-class hepatitis C virus (HCV) nonstructural 5A replication complex inhibitor (NS5A RCI) that is clinically effective in interferon-free combinations with direct-acting antivirals (DAAs) targeting alternate HCV proteins. Recently, we reported NS5A RCI combinations that enhance HCV inhibitory potential in vitro, defining a new class of HCV inhibitors termed NS5A synergists (J. Sun, D. R. O’Boyle II, R. A. Fridell, D. R. Langley, C. Wang, S. Roberts, P. Nower, B. M. Johnson F. Moulin, M. J. Nophsker, Y. Wang, M. Liu, K. Rigat, Y. Tu, P. Hewawasam, J. Kadow, N. A. Meanwell, M. Cockett, J. A. Lemm, M. Kramer, M. Belema, and M. Gao, Nature 527:245–248, 2015, doi:10.1038/nature15711). To extend the characterization of NS5A synergists, we tested new combinations of DCV and NS5A synergists against genotype (gt) 1 to 6 replicons and gt 1a, 2a, and 3a viruses. The kinetics of inhibition in HCV-infected cells treated with DCV, an NS5A synergist (NS5A-Syn), or a combination of DCV and NS5A-Syn were distinctive. Similar to activity observed clinically, DCV caused a multilog drop in HCV, followed by rebound due to the emergence of resistance. DCV–NS5A-Syn combinations were highly efficient at clearing cells of viruses, in line with the trend seen in replicon studies. The retreatment of resistant viruses that emerged using DCV monotherapy with DCV–NS5A-Syn resulted in a multilog drop and rebound in HCV similar to the initial decline and rebound observed with DCV alone on wild-type (WT) virus. A triple combination of DCV, NS5A-Syn, and a DAA targeting the NS3 or NS5B protein cleared the cells of viruses that are highly resistant to DCV. Our data support the observation that the cooperative interaction of DCV and NS5A-Syn potentiates both the genotype coverage and resistance barrier of DCV, offering an additional DAA option for combination therapy and tools for explorations of NS5A function. PMID:26711745

  17. Inhibitors of urokinase type plasminogen activator and cytostatic activity from crude plants extracts.

    PubMed

    Zha, Xueqiang; Diaz, Ricardo; Franco, Jose Javier Rosado; Sanchez, Veronica Forbes; Fasoli, Ezio; Barletta, Gabriel; Carvajal, Augusto; Bansal, Vibha

    2013-01-01

    In view of the clear evidence that urokinase type plasminogen activator (uPA) plays an important role in the processes of tumor cell metastasis, aortic aneurysm, and multiple sclerosis, it has become a target of choice for pharmacological intervention. The goal of this study was thus to determine the presence of inhibitors of uPA in plants known traditionally for their anti-tumor properties. Crude methanol extracts were prepared from the leaves of plants (14) collected from the subtropical dry forest (Guanica, Puerto Rico), and tested for the presence of inhibitors of uPA using the fibrin plate assay. The extracts that tested positive (6) were then partitioned with petroleum ether, chloroform, ethyl acetate and n-butanol, in a sequential manner. The resulting fractions were then tested again using the fibrin plate assay. Extracts from leaves of Croton lucidus (C. lucidus) showed the presence of a strong uPA inhibitory activity. Serial dilutions of these C. lucidus partitions were performed to determine the uPA inhibition IC₅₀ values. The chloroform extract showed the lowest IC₅₀ value (3.52 µg/mL) and hence contained the most potent uPA inhibitor. Further investigations revealed that the crude methanol extract and its chloroform and n-butanol partitions did not significantly inhibit closely related proteases such as the tissue type plasminogen activator (tPA) and plasmin, indicating their selectivity for uPA, and hence superior potential for medicinal use with fewer side effects. In a further evaluation of their therapeutic potential for prevention of cancer metastasis, the C. lucidus extracts displayed cytostatic activity against human pancreatic carcinoma (PaCa-2) cells, as determined through an MTS assay. The cytostatic activities recorded for each of the partitions correlated with their relative uPA inhibitory activities. There are no existing reports of uPA inhibitors being present in any of the plants reported in this study.

  18. Inhibitors of Urokinase Type Plasminogen Activator and Cytostatic Activity from Crude Plants Extracts

    PubMed Central

    Zha, Xueqiang; Diaz, Ricardo; Franco, Jose Javier Rosado; Sanchez, Veronica Forbes; Fasoli, Ezio; Barletta, Gabriel; Carvajal, Augusto; Bansal, Vibha

    2014-01-01

    In view of the clear evidence that urokinase type plasminogen activator (uPA) plays an important role in the processes of tumor cell metastasis, aortic aneurysm, and multiple sclerosis, it has become a target of choice for pharmacological intervention. The goal of this study was thus to determine the presence of inhibitors of uPA in plants known traditionally for their anti-tumor properties. Crude methanol extracts were prepared from the leaves of plants (14) collected from the subtropical dry forest (Guanica, Puerto Rico), and tested for the presence of inhibitors of uPA using the fibrin plate assay. The extracts that tested positive (6) were then partitioned with petroleum ether, chloroform, ethyl acetate and n-butanol, in a sequential manner. The resulting fractions were then tested again using the fibrin plate assay. Extracts from leaves of Croton lucidus (C. lucidus) showed the presence of a strong uPA inhibitory activity. Serial dilutions of these C. lucidus partitions were performed to determine the uPA inhibition IC50 values. The chloroform extract showed the lowest IC50 value (3.52 μg/mL) and hence contained the most potent uPA inhibitor. Further investigations revealed that the crude methanol extract and its chloroform and n-butanol partitions did not significantly inhibit closely related proteases such as the tissue type plasminogen activator (tPA) and plasmin, indicating their selectivity for uPA, and hence superior potential for medicinal use with fewer side effects. In a further evaluation of their therapeutic potential for prevention of cancer metastasis, the C. lucidus extracts displayed cytostatic activity against human pancreatic carcinoma (PaCa-2) cells, as determined through an MTS assay. The cytostatic activities recorded for each of the partitions correlated with their relative uPA inhibitory activities. There are no existing reports of uPA inhibitors being present in any of the plants reported in this study. PMID:23896619

  19. Characterization of Lassa Virus Cell Entry Inhibitors: Determination of the Active Enantiomer by Asymmetric Synthesis

    PubMed Central

    Whitby, Landon R.; Lee, Andrew M.; Kunz, Stefan; Oldstone, Michael B. A.; Boger, Dale L.

    2009-01-01

    The comparative characterization of a series of 4-acyl-1,6-dialkylpiperazin-2-ones as potent cell entry inhibitors of the hemorrhagic fever arenavirus Lassa (LASV) is disclosed. The resolution and examination of the individual enantiomers of the prototypical LASV cell entry inhibitor 3 (16G8) is reported and the more potent (–)-enantiomer was found to be 15-fold more active than the corresponding (+)-enantiomer. The absolute configuration of (–)-3 was established by asymmetric synthesis of the active inhibitor (–)-(S)-3 (lassamycin-1). A limited deletion scan of lassamycin-1 defined key structural features required of the prototypical inhibitors. PMID:19428249

  20. Small molecule inhibitor screen identifies synergistic activity of the bromodomain inhibitor CPI203 and bortezomib in drug resistant myeloma

    PubMed Central

    Siegel, Matthew B.; Liu, Selina Qiuying; Davare, Monika A.; Spurgeon, Stephen E.; Loriaux, Marc M.; Druker, Brian J.

    2015-01-01

    Purpose Despite significant therapeutic progress in multiple myeloma, drug resistance is uniformly inevitable and new treatments are needed. Our aim was to identify novel, efficacious small-molecule combinations for use in drug resistant multiple myeloma. Experimental Design A panel of 116 small molecule inhibitors was used to screen resistant myeloma cell lines for potential therapeutic targets. Agents found to have enhanced activity in the bortezomib or melphalan resistant myeloma cell lines were investigated further in combination. Synergistic combinations of interest were evaluated in primary patient cells. Results The overall single-agent drug sensitivity profiles were dramatically different between melphalan and bortezomib resistant cells, however, the bromodomain inhibitor, CPI203, was observed to have enhanced activity in both the bortezomib and melphalan resistant lines compared to their wild-type counterparts. The combination of bortezomib and CPI203 was found to be synergistic in both the bortezomib and melphalan resistant cell lines as well as in a primary multiple myeloma sample from a patient refractory to recent proteasome inhibitor treatment. The CPI203-bortezomib combination led to enhanced apoptosis and anti-proliferative effects. Finally, in contrast to prior reports of synergy between bortezomib and other epigenetic modifying agents, which implicated MYC downregulation or NOXA induction, our analyses suggest that CPI203-bortezomib synergy is independent of these events. Conclusion Our preclinical data supports a role for the clinical investigation of the bromodomain inhibitor CPI203 combined with bortezomib or alkylating agents in resistant multiple myeloma. PMID:26254279

  1. Effects of inhibitors of tumoricidal activity upon schistosomulum killing by activated macrophages.

    PubMed

    James, S L; Glaven, J A

    1987-12-01

    Larvae of the helminth parasite Schistosoma mansoni are efficiently killed in vitro by lymphokine-activated macrophages, leading to the hypothesis that these cells may participate in the effector mechanism of protective immunity against schistosomiasis. Larvacidal activity has also been demonstrated in the IC-21 macrophage cell line in the absence of a demonstrable respiratory burst, indicating that macrophages possess nonoxidative mechanisms of schistosomulum killing. In this study, we demonstrated that IC-21 larval killing was most effective when contact was allowed between cells and target. Nonoxidative larvacidal activity was prevented by protein synthesis inhibitors, by the inhibition of microtubule polymerization, and by tosyllysylchloromethylketone but not by other inhibitors or substrates of tryptic or chymotryptic protease activity. The addition of excess iron to the culture also prevented IC-21-mediated larval killing, suggesting that the production of an iron-binding molecule may be involved. In contrast, the addition of excess thymidine or arginine did not reverse macrophage larvacidal activity, nor did lysosomotropic agents that depress the activity of acid hydrolases. Under appropriate conditions of activation and surface membrane stimulation, IC-21 cells could be induced to release soluble cytotoxic factors retaining larvacidal activity. These observations provide insight into the mechanism of macrophage-mediated schistosome killing, in comparison to the cytotoxic mechanisms described in the better-studied tumoricidal models, and supply a basis for further biochemical investigation of macrophage function against a multicellular target. PMID:3119500

  2. Effects of inhibitors of tumoricidal activity upon schistosomulum killing by activated macrophages.

    PubMed Central

    James, S L; Glaven, J A

    1987-01-01

    Larvae of the helminth parasite Schistosoma mansoni are efficiently killed in vitro by lymphokine-activated macrophages, leading to the hypothesis that these cells may participate in the effector mechanism of protective immunity against schistosomiasis. Larvacidal activity has also been demonstrated in the IC-21 macrophage cell line in the absence of a demonstrable respiratory burst, indicating that macrophages possess nonoxidative mechanisms of schistosomulum killing. In this study, we demonstrated that IC-21 larval killing was most effective when contact was allowed between cells and target. Nonoxidative larvacidal activity was prevented by protein synthesis inhibitors, by the inhibition of microtubule polymerization, and by tosyllysylchloromethylketone but not by other inhibitors or substrates of tryptic or chymotryptic protease activity. The addition of excess iron to the culture also prevented IC-21-mediated larval killing, suggesting that the production of an iron-binding molecule may be involved. In contrast, the addition of excess thymidine or arginine did not reverse macrophage larvacidal activity, nor did lysosomotropic agents that depress the activity of acid hydrolases. Under appropriate conditions of activation and surface membrane stimulation, IC-21 cells could be induced to release soluble cytotoxic factors retaining larvacidal activity. These observations provide insight into the mechanism of macrophage-mediated schistosome killing, in comparison to the cytotoxic mechanisms described in the better-studied tumoricidal models, and supply a basis for further biochemical investigation of macrophage function against a multicellular target. PMID:3119500

  3. Tissue-type plasminogen activator-plasmin-BDNF modulate glutamate-induced phase-shifts of the mouse suprachiasmatic circadian clock in vitro.

    PubMed

    Mou, Xiang; Peterson, Cynthia B; Prosser, Rebecca A

    2009-10-01

    The mammalian circadian clock in the suprachiasmatic nucleus (SCN) maintains environmental synchrony through light signals transmitted by glutamate released from retinal ganglion terminals. Brain-derived neurotrophic factor (BDNF) is required for light/glutamate to reset the clock. In the hippocampus, BDNF is activated by the extracellular protease, plasmin, which is produced from plasminogen by tissue-type plasminogen activator (tPA). We provide data showing expression of proteins from the plasminogen activation cascade in the SCN and their involvement in circadian clock phase-resetting. Early night glutamate application to SCN-containing brain slices resets the circadian clock. Plasminogen activator inhibitor-1 (PAI-1) blocked these shifts in slices from wild-type mice but not mice lacking its stabilizing protein, vitronectin (VN). Plasmin, but not plasminogen, prevented inhibition by PAI-1. Both plasmin and active BDNF reversed alpha(2)-antiplasmin inhibition of glutamate-induced shifts. alpha(2)-Antiplasmin decreased the conversion of inactive to active BDNF in the SCN. Finally, both tPA and BDNF allowed daytime glutamate-induced phase-resetting. Together, these data are the first to demonstrate expression of these proteases in the SCN, their involvement in modulating photic phase-shifts, and their activation of BDNF in the SCN, a potential 'gating' mechanism for photic phase-resetting. These data also demonstrate a functional interaction between PAI-1 and VN in adult brain. Given the usual association of these proteins with the extracellular matrix, these data suggest new lines of investigation into the locations and processes modulating mammalian circadian clock phase-resetting.

  4. Localization and regulation of the tissue plasminogen activator-plasmin system in the hippocampus.

    PubMed

    Salles, Fernando J; Strickland, Sidney

    2002-03-15

    The extracellular protease cascade of tissue plasminogen activator (tPA) and plasminogen has been implicated in neuronal plasticity and degeneration. We show here that unstimulated expression of tPA in the mouse hippocampus is concentrated in the mossy fiber pathway, with little or no expression within the perforant path, the Schaffer collaterals, or neuronal cell bodies. tPA protein is also expressed in vascular endothelial cells throughout the brain parenchyma. Four hours after excitotoxic injury, tPA protein is transiently induced within CA1 pyramidal neurons. The induced CA1 tPA is localized to neurons that survive the injury and is enzymatically active. Within the mossy fiber pathway, injury resulted in decreased tPA protein. In contrast, mossy fiber tPA activity displayed a biphasic character: transient increase at 8 hr, then a decrease by 24 hr after injury. Analysis of plasminogen activator inhibitor-1 (PAI-1) expression showed that PAI-1 antigen is upregulated by 24 hr and could account for the tPA activity downregulation seen at this time point. Plasminogen immunohistochemistry suggested an increase within the mossy fiber pathway after injury. Finally, hippocampal tPA expression among various mammalian species was strikingly different. These results indicate a complex control of tPA protein and enzymatic activity in the hippocampus that may help regulate neuronal plasticity.

  5. A thermostable trypsin inhibitor with antiproliferative activity from small pinto beans.

    PubMed

    Chan, Yau Sang; Zhang, Yanbo; Sze, Stephen Cho Wing; Ng, Tzi Bun

    2014-08-01

    Small pinto bean is a cultivar of Phaseolus vulgaris. It produces a 16-kDa trypsin inhibitor that could be purified using anion exchange and size chromatography. Q-Sepharose, Mono Q and Superdex 75 columns were employed for the isolation process. Small pinto bean trypsin inhibitor demonstrated moderate pH stability (pH 2-10) and marked heat stability, with its trypsin inhibitory activity largely retained after exposure to 100 °C for half an hour. The activity was abolished in the presence of dithiothreitol, in a dose-dependent manner, implying that disulfide bonds in small pinto bean trypsin inhibitor are crucial for the activity. The trypsin inhibitor showed a blocked N-terminus. The trypsin inhibitor only slightly inhibited the viability of breast cancer MCF7 and hepatoma HepG2 cells at 125 μM. PMID:23859150

  6. Antiviral activity of a Rac GEF inhibitor characterized with a sensitive HIV/SIV fusion assay

    SciTech Connect

    Pontow, Suzanne; Harmon, Brooke; Campbell, Nancy; Ratner, Lee

    2007-11-10

    A virus-dependent fusion assay was utilized to examine the activity of a panel of HIV-1, -2, and SIV isolates of distinct coreceptor phenotypes. This assay allowed identification of entry inhibitors, and characterization of an antagonist of a Rac guanine nucleotide exchange factor, as an inhibitor of HIV-mediated fusion.

  7. The activity and location of cathepsin D inhibitor in seeds of common vetch (Vicia sativa L.).

    PubMed

    Roszkowska-Jakimiec, W; Leśniewska, J

    2004-01-01

    The activity of cathepsin D inhibitor is markedly higher in common vetch seed coat than in embryo cotyledons. The occurrence of considerable amounts of the inhibitor in the seed coat of vetch was confirmed by the fluorescent microscopic technique, with the use of fluorescein-marked cathepsin D.

  8. A nonnucleoside reverse transcriptase inhibitor active on human immunodeficiency virus type 1 isolates resistant to related inhibitors.

    PubMed Central

    Goldman, M E; O'Brien, J A; Ruffing, T L; Schleif, W A; Sardana, V V; Byrnes, V W; Condra, J H; Hoffman, J M; Emini, E A

    1993-01-01

    Pyridinone derivatives are potent and specific inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and HIV-1 replication in cell culture. However, the potential clinical usefulness of these compounds as monotherapeutic agents may be limited by the selection of inhibitor-resistant viral variants. Resistance in cell culture is due primarily to mutational alterations at RT amino acid residues 103 and 181. A recombinant HIV-1 RT containing both of these mutations was used to screen a panel of pyridinone analogs for inhibitory activity. L-696,229 and L-697,661, pyridinones currently undergoing clinical evaluation, were more than 4,000-fold weaker against the mutant enzyme than against the wild-type enzyme. In contrast, one derivative of L-696,229, L-702,019 (3-[2-(4,7-dichlorobenzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyrid in-2(1H)-thione), showed only three-fold different potencies against the two enzymes. L-702,019 was also a potent inhibitor of the replication of mutant HIV-1 containing the individual mutations at amino acid 103 or 181 as well as of clinical isolates resistant to L-697,661 and L-696,229. Isolation and analysis of resistant viral variants in cell culture showed that significant resistance to L-702,019 could be engendered only by multiple amino acid substitutions in RT. Accordingly, these studies demonstrated the potential of identifying second-generation specific HIV-1 RT inhibitors that can overcome the viral resistance selected by the first generation of inhibitors. PMID:7685996

  9. Discovery of HDAC Inhibitors with Potent Activity Against Multiple Malaria Parasite Life Cycle Stages

    PubMed Central

    Hansen, Finn K.; Sumanadasa, Subathdrage D. M.; Stenzel, Katharina; Duffy, Sandra; Meister, Stephan; Marek, Linda; Schmetter, Rebekka; Kuna, Krystina; Hamacher, Alexandra; Mordmüller, Benjamin; Kassack, Matthias U.; Winzeler, Elizabeth A.; Avery, Vicky M.; Andrews, Katherine T.; Kurz, Thomas

    2015-01-01

    In this work we investigated the antiplasmodial activity of a series of HDAC inhibitors containing an alkoxyamide connecting-unit linker region. HDAC inhibitor 1a (LMK235), previously shown to be a novel and specific inhibitor of human HDAC4 and 5, was used as a starting point to rapidly construct a mini-library of HDAC inhibitors using a straightforward solid-phase supported synthesis. Several of these novel HDAC inhibitors were found to have potent in vitro activity against asexual stage P. falciparum malaria parasites. Representative compounds were shown to hyperacetylate P. falciparum histones and to inhibit deacetylase activity of recombinant PfHDAC1 and P. falciparum nuclear extracts. All compounds were also screened in vitro for activity against P. berghei exo-erythrocytic stages and selected compounds were further tested against late stage (IV and V) P. falciparum gametocytes. Of note, some compounds showed nanomolar activity against all three life cycle stages tested (asexual, exo-erythrocytic and gametocyte stages) and several compounds displayed significantly increased parasite selectivity compared to the reference HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). These data suggest that it may be possible to develop HDAC inhibitors that target multiple malaria parasite life cycle stages. PMID:24904967

  10. Identification of quinazolinyloxy biaryl urea as a new class of SUMO activating enzyme 1 inhibitors.

    PubMed

    Kumar, Ashutosh; Ito, Akihiro; Hirohama, Mikako; Yoshida, Minoru; Zhang, Kam Y J

    2013-09-15

    SUMO activating enzyme 1 (SUMO E1) is the first enzyme in sumoylation pathway and an important cancer drug target. However, only a few inhibitors were reported up to now that includes three natural products, semi-synthetic protein inhibitors and one AMP mimic. Here, we report the identification of quinazolinyloxy biaryl urea as a new class of SUMO E1 inhibitors. The most active compound of this class inhibited the in vitro sumoylation with an IC50 of 13.4 μM. This compound inhibits sumoylation by blocking the formation of SUMOE1-SUMO thioester intermediate. The biological activity of the most active compound is comparable to previously reported inhibitors with properties suitable for medicinal chemistry optimization for potency and druggability.

  11. Pattern formation in a (2 + 1)-species activator-inhibitor-immobilizer system

    NASA Astrophysics Data System (ADS)

    Pearson, John E.

    1992-09-01

    The necessary and sufficient conditions for a Turing instability in a (2 + 1)-component reaction-diffusion system are derived. The 2-component subsystem consists of an arbitrary activator-inhibitor system in which the activator and the inhibitor diffuse at identical rates. The activator reacts with an immobile substrate to form an immobile complex which does not diffuse. It is found that the critical wavenumber and the location of the instability in parameter space are independent of the initial substrate concentration. As a special case, the (2 + 1)-variable activator-inhibitor-immobilizer system can be reduced to an activator-inhibitor system in which the activator diffuses more slowly than the inhibitor. This occurs when the time scales in the system satisfy certain constraints. The general results are applied to a (2 + 1)-variable model of the chlorite-iodide-starch reaction recently proposed by Epstein and Lengyel to explain the experimental observation of Turing patterns in a related system. I find an intrinsic wavelength which is within a factor of two of the experimentally observed wavelength, but the necessary conditions for the (2 + 1)-variable system to reduce to a two-variable activator-inhibitor system with rescaled diffusion coefficients are not satisfied.

  12. The Appearance of New Active Forms of Trypsin Inhibitor in Germinating Mung Bean (Vigna radiata) Seeds.

    PubMed

    Lorensen, E; Prevosto, R; Wilson, K A

    1981-07-01

    Ungerminated seeds of mung bean contain a single major species (F) of trypsin inhibitor with five minor species (A-E) separable on diethylaminoethyl-cellulose. During germination the level of trypsin inhibitory activity decreases from 1.8 units/grams dry weight in ungerminated cotyledons to 1.2 units/grams in cotyledons from seeds germinated 5 days. This decrease is accompanied by major changes in the distribution of inhibitory activity among the inhibitor species. By 48 hours of germination, inhibitor F has largely disappeared with an accompanying rapid increase in inhibitor C. Similarly, though less rapidly, inhibitor E decreases while inhibitor A increases. A similar sequence of changes is found in vitro when purified inhibitor F is incubated with extracts from seeds germinated 96 hours. The combined in vivo and in vitro data suggest a conversion sequence of: F --> E --> C --> A. The in vitro conversion is inhibited by phenylmethyl sulfonyl fluoride but not by iodoacetamide, indicating that at least the initial phases of inhibitor conversion are not catalyzed by the mung bean vicilin peptidohydrolase.

  13. Structure-based repurposing of FDA-approved drugs as inhibitors of NEDD8-activating enzyme.

    PubMed

    Zhong, Hai-Jing; Liu, Li-Juan; Chan, Daniel Shiu-Hin; Wang, Hui-Min; Chan, Philip Wai Hong; Ma, Dik-Lung; Leung, Chung-Hang

    2014-07-01

    We report the discovery of an inhibitor of NEDD8-activating enzyme (NAE) by an integrated virtual screening approach. Piperacillin 1 inhibited NAE activity in cell-free and cell-based systems with high selectivity. Furthermore, piperacillin 1 was able to inhibit the degradation of the NAE downstream protein substrate p27(kip1). Our molecular modeling and kinetic studies suggested that this compound may act as a non-covalent ATP-competitive inhibitor of NAE.

  14. Immunomodulatory activity of a chymotrypsin inhibitor from Momordica cochinchinensis seeds.

    PubMed

    Tsoi, Alex Yuen-Kam; Ng, Tzi-Bun; Fong, Wing-Ping

    2006-09-01

    Serine protease inhibitors are widely distributed in the plant kingdom. Many of them have been purified and characterized from different species. While the physicochemical properties of these protease inhibitors have been extensively investigated, their biological effects, e.g. immunomodulatory effect, remain relatively unexplored. Recently, we isolated a chymotrypsin-specific inhibitor (MCoCI) from the seeds of Momordica cochinchinensis (Lour) Spreng (Family Cucurbitaceae), the traditional Chinese medicine known as Mubiezhi, which has been used as an antiinflammatory agent. In the present study, the effects of MCoCI on different types of cells of the immune system, including splenocytes, splenic lymphocytes, neutrophils, bone marrow cells and macrophages, were investigated. MCoCI was shown to possess immuno-enhancing and antiinflammatory effects. MCoCI could stimulate the proliferation of different cells of the immune system, e.g. splenocytes, splenic lymphocytes and bone marrow cells, in a manner comparable to that of Concanavalin A. Moreover, MCoCI could also suppress the formation of hydrogen peroxide in neutrophils and macrophages. These immunomodulatory effects may explain some of the therapeutic actions of Mubiezhi. PMID:16733830

  15. Immunomodulatory activity of a chymotrypsin inhibitor from Momordica cochinchinensis seeds.

    PubMed

    Tsoi, Alex Yuen-Kam; Ng, Tzi-Bun; Fong, Wing-Ping

    2006-09-01

    Serine protease inhibitors are widely distributed in the plant kingdom. Many of them have been purified and characterized from different species. While the physicochemical properties of these protease inhibitors have been extensively investigated, their biological effects, e.g. immunomodulatory effect, remain relatively unexplored. Recently, we isolated a chymotrypsin-specific inhibitor (MCoCI) from the seeds of Momordica cochinchinensis (Lour) Spreng (Family Cucurbitaceae), the traditional Chinese medicine known as Mubiezhi, which has been used as an antiinflammatory agent. In the present study, the effects of MCoCI on different types of cells of the immune system, including splenocytes, splenic lymphocytes, neutrophils, bone marrow cells and macrophages, were investigated. MCoCI was shown to possess immuno-enhancing and antiinflammatory effects. MCoCI could stimulate the proliferation of different cells of the immune system, e.g. splenocytes, splenic lymphocytes and bone marrow cells, in a manner comparable to that of Concanavalin A. Moreover, MCoCI could also suppress the formation of hydrogen peroxide in neutrophils and macrophages. These immunomodulatory effects may explain some of the therapeutic actions of Mubiezhi.

  16. p53 Small Molecule Inhibitor Enhances Temozolomide Cytotoxic Activity against Intracranial Glioblastoma Xenografts

    PubMed Central

    Dinca, Eduard B.; Lu, Kan V.; Sarkaria, Jann N.; Pieper, Russell O.; Prados, Michael D.; Haas-Kogan, Daphne A.; VandenBerg, Scott R.; Berger, Mitchel S.; James, C. David

    2010-01-01

    In this study we investigated corresponding precursor and active forms of a p53 small molecule inhibitor for effect on temozolomide (TMZ) anti-tumor activity against glioblastoma (GBM), using both in vitro and in vivo experimental approaches. Results from in vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased when GBMs with wild-type p53 were co-treated with the active form of p53 inhibitor, and this heightened cytotoxic response was accompanied by increased PARP cleavage as well as elevated cellular phospho-H2AX. Analysis of the same series of GBMs, as intracranial xenografts in athymic mice, and administering corresponding p53 inhibitor precursor, that is converted to the active compound in vivo, yielded results consistent with the in vitro analyses: i.e., TMZ + p53 inhibitor precursor co-treatment, of three distinct wild-type p53 GBM xenografts, resulted in significant enhancement of TMZ anti-tumor effect relative to treatment with TMZ alone, as indicated by serial bioluminescence monitoring as well as survival analysis (p < 0.001 for co-treatment survival benefit in each case). Mice receiving intracranial injection with p53 null GBM showed similar survival benefit from TMZ treatment regardless of the presence or absence of p53 inhibitor precursor. In total, our results indicate that the p53 active and precursor inhibitor pair enhance TMZ cytotoxicity in vitro and in vivo, respectively, and do so in a p53-dependent manner. PMID:19074867

  17. p53 Small-molecule inhibitor enhances temozolomide cytotoxic activity against intracranial glioblastoma xenografts.

    PubMed

    Dinca, Eduard B; Lu, Kan V; Sarkaria, Jann N; Pieper, Russell O; Prados, Michael D; Haas-Kogan, Daphne A; Vandenberg, Scott R; Berger, Mitchel S; James, C David

    2008-12-15

    In this study, we investigated the precursor and active forms of a p53 small-molecule inhibitor for their effects on temozolomide (TMZ) antitumor activity against glioblastoma (GBM), using both in vitro and in vivo experimental approaches. Results from in vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased when p53 wild-type (p53(wt)) GBMs were cotreated with the active form of p53 inhibitor, and this heightened cytotoxic response was accompanied by increased poly(ADP-ribose) polymerase cleavage as well as elevated cellular phospho-H2AX. Analysis of the same series of GBMs, as intracranial xenografts in athymic mice, and administering corresponding p53 inhibitor precursor, which is converted to the active compound in vivo, yielded results consistent with the in vitro analyses: TMZ + p53 inhibitor precursor cotreatment of three distinct p53(wt) GBM xenografts resulted in significant enhancement of TMZ antitumor effect relative to treatment with TMZ alone, as indicated by serial bioluminescence monitoring as well as survival analysis (P < 0.001 for cotreatment survival benefit in each case). Mice receiving intracranial injection with p53(null) GBM showed similar survival benefit from TMZ treatment regardless of the presence or absence of p53 inhibitor precursor. In total, our results indicate that the p53 active and precursor inhibitor pair enhances TMZ cytotoxicity in vitro and in vivo, respectively, and do so in a p53-dependent manner.

  18. Protease inhibitor in scorpion (Mesobuthus eupeus) venom prolongs the biological activities of the crude venom.

    PubMed

    Ma, Hakim; Xiao-Peng, Tang; Yang, Shi-Long; Lu, Qiu-Min; Lai, Ren

    2016-08-01

    It is hypothesized that protease inhibitors play an essential role in survival of venomous animals through protecting peptide/protein toxins from degradation by proteases in their prey or predators. However, the biological function of protease inhibitors in scorpion venoms remains unknown. In the present study, a trypsin inhibitor was purified and characterized from the venom of scorpion Mesobuthus eupeus, which enhanced the biological activities of crude venom components in mice when injected in combination with crude venom. This protease inhibitor, named MeKTT-1, belonged to Kunitz-type toxins subfamily. Native MeKTT-1 selectively inhibited trypsin with a Kivalue of 130 nmol·L(-1). Furthermore, MeKTT-1 was shown to be a thermo-stable peptide. In animal behavioral tests, MeKTT-1 prolonged the pain behavior induced by scorpion crude venom, suggesting that protease inhibitors in scorpion venom inhibited proteases and protect the functionally important peptide/protein toxins from degradation, consequently keeping them active longer. In conclusion, this was the first experimental evidence about the natural existence of serine protease inhibitor in the venom of scorpion Mesobuthus eupeus, which preserved the activity of venom components, suggests that scorpions may use protease inhibitors for survival. PMID:27608950

  19. Detection of allosteric kinase inhibitors by displacement of active site probes.

    PubMed

    Lebakken, Connie S; Reichling, Laurie J; Ellefson, Jason M; Riddle, Steven M

    2012-07-01

    Non-adenosine triphosphate (ATP) competitive, allosteric inhibitors provide a promising avenue to develop highly selective small-molecule kinase inhibitors. Although this class of compounds is growing, detection of such inhibitors can be challenging as standard kinase activity assays preferentially detect compounds that bind to active kinases in an ATP competitive manner. We have previously described a time-resolved fluorescence resonance energy transfer (TR-FRET)-based kinase binding assay using the competitive displacement of ATP competitive active site fluorescent probes ("tracers"). Although this format has gained acceptance, published data with this and related formats are almost entirely without examples of non-ATP competitive compounds. Thus, this study addresses whether this format is useful for non-ATP competitive inhibitors. To this end, 15 commercially available non-ATP competitive inhibitors were tested for their ability to displace ATP competitive probes. Despite the diversity of both compound structures and their respective targets, 14 of the 15 compounds displaced the tracers with IC(50) values comparable to literature values. We conclude that such binding assays are well suited for the study of non-ATP competitive inhibitors. In addition, we demonstrate that allosteric inhibitors of BCR-Abl and MEK bind preferentially to the nonphosphorylated (i.e., inactive) form of the kinase, indicating that binding assays may be a preferred format in some cases.

  20. Development of an activity assay for discovery of inhibitors of lipopolysaccharide transport.

    PubMed

    Gronenberg, Luisa S; Kahne, Daniel

    2010-03-01

    The outer membrane of gram-negative bacteria contains an outer leaflet composed of lipopolysaccharide (LPS) that is transported to this location by a pathway that is essential for viability. It has been suggested that inhibitors of this pathway could be useful antibiotics. Herein we reconstitute the activity of the ATPase component (LptB) of the ABC transporter that initiates LPS transport and assembly. We developed a high-throughput assay and screened a library of kinase inhibitors against LptB. We identified two classes of ATP-competitive inhibitors. These are the first inhibitors of the ATPase component of any bacterial ABC transporter. The small-molecule inhibitors will be very useful tools for further biochemical studies of the proteins involved in LPS transport and assembly.

  1. Structure-activity relationships by mass spectrometry: identification of novel MMP-3 inhibitors.

    PubMed

    Ockey, Denise A; Dotson, Jenna L; Struble, Martin E; Stults, John T; Bourell, James H; Clark, Kevin R; Gadek, Thomas R

    2004-01-01

    A novel class of nonpeptide inhibitors of stromelysin (MMP-3) has been discovered with the use of mass spectrometry. The method relies on the development of structure-activity relationships by mass spectrometry (SAR by MS) and utilizes information derived from the binding of known inhibitors to identify novel inhibitors of a target protein with a minimum of synthetic effort. Noncovalent complexes of known inhibitors with a target protein are analyzed; these inhibitors are deconstructed into sets of fragments which compete for common or overlapping binding sites on the target protein. The binding of each fragment set can be studied independently. With the use of competition studies, novel members of each fragment set are identified from compound libraries that bind to the same site on the target protein. A novel inhibitor of the target protein was then constructed by chemically linking a combination of members of each fragment set in a manner guided by the proximity and orientation of the fragments derived from the known inhibitors. In the case of stromelysin, a novel inhibitor composed of favorably linked fragments was observed to form a 1:1 complex with stromelysin. Compounds that were not linked appropriately formed higher order complexes with stoichiometries of 2:1 or greater. These linked molecules were subsequently assessed for their ability to block stromelysin function in a chromogenic substrate assay.

  2. Inflammatory Gene Expression Upon TGF-β1-Induced p38 Activation in Primary Dupuytren's Disease Fibroblasts

    PubMed Central

    Bujak, Maro; Ratkaj, Ivana; Markova-Car, Elitza; Jurišić, Davor; Horvatić, Anita; Vučinić, Srđan; Lerga, Jonatan; Baus-Lončar, Mirela; Pavelić, Krešimir; Kraljević Pavelić, Sandra

    2015-01-01

    Objectives: Inflammation is an underlying mechanism behind fibrotic processes and differentiation of cells into myofibroblasts. Presented study therefore provides new data on activation of autoimmune and inflammatory immune response genes that accompany activation of p38 and cell differentiation in primary cells derived from Dupuytren's disease (DD) patients. Methods: Primary non-Dupuytren's disease cells (ND) were isolated from macroscopically unaffected palmar fascia adjacent to diseased tissue obtained from patients diagnosed with the last stage of DD and cultured in vitro. Gene expression, collagen gel contraction assay and analysis of secreted proteins were performed in ND cells treated with TGF-β1 and/or inhibitor of p38 phosphorylation. Results: During differentiation of ND fibroblasts, increased expression of immune response genes PAI-1, TIMP-1, CCL11, and IL-6 was found. These changes were accompanied by increased cell contractility and activation of p38 and its target kinase MK2. Inhibition of p38 phosphorylation reversed these processes in vitro. Conclusions: TGF-β1 induced p38 phosphorylation in ND cells grown from macroscopically unaffected palmar fascia adjacent to diseased tissue from DD patients. This was accompanied by activation of the cytokine genes CCL-11 and IL-6 and secretion of extracellular matrix regulatory proteins PAI-1 and TIMP-1. A combined approach directed toward inflammation and p38 MAPK-mediated processes in DD might be considered for improving management of DD patients and prevention of recurrence. PMID:26697433

  3. Effects of the contraceptive skin patch and subdermal contraceptive implant on markers of endothelial cell activation and inflammation.

    PubMed

    Hernandez-Juarez, Jesus; Sanchez-Serrano, Juan Carlos; Moreno-Hernandez, Manuel; Alvarado-Moreno, Jose Antonio; Hernandez-Lopez, Jose Rubicel; Isordia-Salas, Irma; Majluf-Cruz, Abraham

    2015-07-01

    Changes in blood coagulation factors may partially explain the association between hormonal contraceptives and thrombosis. Therefore, the likely effects of the contraceptive skin patch and subdermal contraceptive implant on levels of inflammatory markers and endothelial activation were analyzed. This was an observational, prospective, longitudinal, nonrandomized study composed of 80 women between 18 and 35 years of age who made the decision to use the contraceptive skin patch or subdermal contraceptive implant. vascular cell adhesion molecule-1 (VCAM-1), endothelial cell leukocyte adhesion molecule-1 (ELAM-1), von Willebrand factor (VWF), and plasminogen activator inhibitor type 1(PAI-1) as well as high-sensitivity C-reactive protein (hsCRP) were assayed before and after 4 months of use of the contraceptive method. VCAM-1, VWF, and PAI-1 remained unchanged in the contraceptive skin patch group; however, a significant increase in hsCRP (0.29-0.50 mg/dL; P =.012) and a significant decrease in ELAM-1 (44-25 ng/mL; P =.022) were observed. A significant diminution in VCAM-1 (463-362 ng/mL; P =.022) was also found in the subdermal contraceptive implant group. Our results strongly suggest that these contraceptive methods do not induce endothelial activation after 4 months of use. Increase in hsCRP levels was unrelated to changes in markers of endothelial activation.

  4. PTP1B inhibitor promotes endothelial cell motility by activating the DOCK180/Rac1 pathway

    PubMed Central

    Wang, Yuan; Yan, Feng; Ye, Qing; Wu, Xiao; Jiang, Fan

    2016-01-01

    Promoting endothelial cell (EC) migration is important not only for therapeutic angiogenesis, but also for accelerating re-endothelialization after vessel injury. Several recent studies have shown that inhibition of protein tyrosine phosphatase 1B (PTP1B) may promote EC migration and angiogenesis by enhancing the vascular endothelial growth factor receptor-2 (VEGFR2) signalling. In the present study, we demonstrated that PTP1B inhibitor could promote EC adhesion, spreading and migration, which were abolished by the inhibitor of Rac1 but not RhoA GTPase. PTP1B inhibitor significantly increased phosphorylation of p130Cas, and the interactions among p130Cas, Crk and DOCK180; whereas the phosphorylation levels of focal adhesion kinase, Src, paxillin, or Vav2 were unchanged. Gene silencing of DOCK180, but not Vav2, abrogated the effects of PTP1B inhibitor on EC motility. The effects of PTP1B inhibitor on EC motility and p130Cas/DOCK180 activation persisted in the presence of the VEGFR2 antagonist. In conclusion, we suggest that stimulation of the DOCK180 pathway represents an alternative mechanism of PTP1B inhibitor-stimulated EC motility, which does not require concomitant VEGFR2 activation as a prerequisite. Therefore, PTP1B inhibitor may be a useful therapeutic strategy for promoting EC migration in cardiovascular patients in which the VEGF/VEGFR functions are compromised. PMID:27052191

  5. Tricyclic Covalent Inhibitors Selectively Target Jak3 through an Active Site Thiol*

    PubMed Central

    Goedken, Eric R.; Argiriadi, Maria A.; Banach, David L.; Fiamengo, Bryan A.; Foley, Sage E.; Frank, Kristine E.; George, Jonathan S.; Harris, Christopher M.; Hobson, Adrian D.; Ihle, David C.; Marcotte, Douglas; Merta, Philip J.; Michalak, Mark E.; Murdock, Sara E.; Tomlinson, Medha J.; Voss, Jeffrey W.

    2015-01-01

    The action of Janus kinases (JAKs) is required for multiple cytokine signaling pathways, and as such, JAK inhibitors hold promise for treatment of autoimmune disorders, including rheumatoid arthritis, inflammatory bowel disease, and psoriasis. However, due to high similarity in the active sites of the four members (Jak1, Jak2, Jak3, and Tyk2), developing selective inhibitors within this family is challenging. We have designed and characterized substituted, tricyclic Jak3 inhibitors that selectively avoid inhibition of the other JAKs. This is accomplished through a covalent interaction between an inhibitor containing a terminal electrophile and an active site cysteine (Cys-909). We found that these ATP competitive compounds are irreversible inhibitors of Jak3 enzyme activity in vitro. They possess high selectivity against other kinases and can potently (IC50 < 100 nm) inhibit Jak3 activity in cell-based assays. These results suggest irreversible inhibitors of this class may be useful selective agents, both as tools to probe Jak3 biology and potentially as therapies for autoimmune diseases. PMID:25552479

  6. Targeting the RAS pathway by mitogen-activated protein kinase inhibitors.

    PubMed

    Kiessling, Michael K; Rogler, Gerhard

    2015-01-01

    Targeting of oncogenic driver mutations with small-molecule inhibitors resulted in powerful treatment options for cancer patients in recent years. The RAS (rat sarcoma) pathway is among the most frequently mutated pathways in human cancer. Whereas targeting mutant Kirsten RAS (KRAS) remains difficult, mutant B rapidly accelerated fibrosarcoma (BRAF) kinase is an established drug target in cancer. Now data show that neuroblastoma RAS (NRAS) and even Harvey RAS (HRAS) mutations could be predictive markers for treatment with mitogen-activated protein kinase (MEK) inhibitors. This review discusses recent preclinical and clinical studies of MEK inhibitors in BRAF and RAS mutant cancer. PMID:26691679

  7. Inhibitors of enzymes catalyzing modifications to histone lysine residues: structure, function and activity.

    PubMed

    Lillico, Ryan; Stesco, Nicholas; Khorshid Amhad, Tina; Cortes, Claudia; Namaka, Mike P; Lakowski, Ted M

    2016-05-01

    Gene expression is partly controlled by epigenetic mechanisms including histone-modifying enzymes. Some diseases are caused by changes in gene expression that can be mitigated by inhibiting histone-modifying enzymes. This review covers the enzyme inhibitors targeting histone lysine modifications. We summarize the enzymatic mechanisms of histone lysine acetylation, deacetylation, methylation and demethylation and discuss the biochemical roles of these modifications in gene expression and in disease. We discuss inhibitors of lysine acetylation, deacetylation, methylation and demethylation defining their structure-activity relationships and their potential mechanisms. We show that there are potentially indiscriminant off-target effects on gene expression even with the use of selective epigenetic enzyme inhibitors.

  8. Proteasome inhibitors reduce luciferase and beta-galactosidase activity in tissue culture cells.

    PubMed

    Deroo, Bonnie J; Archer, Trevor K

    2002-06-01

    Reporter enzymes are commonly used in cell biology to study transcriptional activity of genes. Recently, reporter enzymes in combination with compounds that inhibit proteasome function have been used to study the effect of blocking transcription factor degradation on gene activation. While investigating the effect of proteasome inhibition on steroid receptor activation of the mouse mammary tumor virus (MMTV) promoter, we found that treatment with proteasome inhibitors enhanced glucocorticoid activation of the promoter attached to a chloramphenicol acetyltransferase (CAT) reporter, but inhibited activation of MMTV attached to a firefly luciferase or beta-galactosidase reporter. MMTV RNA levels under these conditions correlated with the promoter activity observed using the CAT reporter, suggesting that proteasome inhibitor treatment interfered with luciferase or beta-galactosidase reporter assays. Washout experiments demonstrated that the majority of luciferase activity was lost if the proteasome inhibitor was added at the same time luciferase was produced, not once the functional protein was made, suggesting that proteasome inhibition interferes with production of luciferase protein. Indeed, we found that proteasome inhibitor treatment dramatically reduced the levels of luciferase and beta-galactosidase protein produced, as determined by Western blot. Thus, treatment with proteasome inhibitors interferes with luciferase and beta-galactosidase reporter assays, possibly by inhibiting production of a functional reporter protein.

  9. A straightforward ninhydrin-based method for collagenase activity and inhibitor screening of collagenase using spectrophotometry.

    PubMed

    Zhang, Yanfang; Fu, Yun; Zhou, Sufeng; Kang, Lixia; Li, Changzheng

    2013-06-01

    Currently protease assay kits, requiring substrate that is either radiolabeled or fluorescence labeled and specialized instruments, are all expensive. A simple, reliable assay of protease activity and its inhibitor screening for general laboratory is rare. Here we demonstrated a straightforward ninhydrin-based method for assay of collagenase activity and its inhibitor screening using spectrophotometry. In the method, without multistep sample treatments and substrate labeling, the hydrolytic products were directly traced by ninhydrin. The method is expected to be suitable for not only the assay of collagenase activity but also the others matrix metalloproteinases activities, and can be used for kinetic study.

  10. Aucubin, a naturally occurring iridoid glycoside inhibits TNF-α-induced inflammatory responses through suppression of NF-κB activation in 3T3-L1 adipocytes.

    PubMed

    Park, Kyoung Sik

    2013-06-01

    Obesity is closely associated with a state of chronic, low-grade inflammation characterized by abnormal cytokine production and activation of inflammatory signaling pathways in adipose tissue. Tumor necrosis factor (TNF)-α is chronically elevated in adipose tissues of obese rodents and humans. Increased levels of TNF-α are implicated in the induction of atherogenic adipokines, such as plasminogen activator inhibitor (PAI)-1, adipose-tissue-derived monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6. Aucubin, an iridoid glycoside existing in medicinal plants, has been reported to show an anti-inflammatory activity by suppression of TNF-α production in murine macrophages. The present study is aimed to investigate the effects of aucubin on TNF-α-induced atherogenic changes of the adipokines in differentiated 3T3-L1 cells. Aucubin significantly inhibited TNF-α-induced secretion and mRNA synthesis of the atherogenic adipokines including PAI-1, MCP-1, and IL-6. Further investigation of the molecular mechanism revealed that pretreatment with aucubin suppressed extracellular signal-regulated kinase (ERK) activation, inhibitory kappa Bα (IκBα) degradation, and subsequent nuclear factor kappa B (NF-κB) activation. These findings suggest that aucubin may improve obesity-induced atherosclerosis by attenuating TNF-α-induced inflammatory responses.

  11. Feedback Activation of Leukemia Inhibitory Factor Receptor Limits Response to Histone Deacetylase Inhibitors in Breast Cancer.

    PubMed

    Zeng, Hanlin; Qu, Jia; Jin, Nan; Xu, Jun; Lin, Chenchu; Chen, Yi; Yang, Xinying; He, Xiang; Tang, Shuai; Lan, Xiaojing; Yang, Xiaotong; Chen, Ziqi; Huang, Min; Ding, Jian; Geng, Meiyu

    2016-09-12

    Histone deacetylase (HDAC) inhibitors have demonstrated clinical benefits in subtypes of hematological malignancies. However, the efficacy of HDAC inhibitors in solid tumors remains uncertain. This study takes breast cancer as a model to understand mechanisms accounting for limited response of HDAC inhibitors in solid tumors and to seek combination solutions. We discover that feedback activation of leukemia inhibitory factor receptor (LIFR) signaling in breast cancer limits the response to HDAC inhibition. Mechanistically, HDAC inhibition increases histone acetylation at the LIFR gene promoter, which recruits bromodomain protein BRD4, upregulates LIFR expression, and activates JAK1-STAT3 signaling. Importantly, JAK1 or BRD4 inhibition sensitizes breast cancer to HDAC inhibitors, implicating combination inhibition of HDAC with JAK1 or BRD4 as potential therapies for breast cancer. PMID:27622335

  12. Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site*S

    PubMed Central

    Maresso, Anthony W.; Wu, Ruiying; Kern, Justin W.; Zhang, Rongguang; Janik, Dorota; Missiakas, Dominique M.; Duban, Mark-Eugene; Joachimiak, Andrzej; Schneewind, Olaf

    2011-01-01

    Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of sortase leads to large reductions in virulence, which identifies sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (β-amino)ethyl ketones inhibit sortase enzymes from staphylococci and bacilli. Inhibition of sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via β-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery. PMID:17545669

  13. Modulation of activation-loop phosphorylation by JAK inhibitors is binding mode dependent

    PubMed Central

    Bonenfant, Débora; Rubert, Joëlle; Vangrevelinghe, Eric; Scheufler, Clemens; Marque, Fanny; Régnier, Catherine H.; De Pover, Alain; Ryckelynck, Hugues; Bhagwat, Neha; Koppikar, Priya; Goel, Aviva; Wyder, Lorenza; Tavares, Gisele; Baffert, Fabienne; Pissot-Soldermann, Carole; Manley, Paul W.; Gaul, Christoph; Voshol, Hans; Levine, Ross L.; Sellers, William R.; Hofmann, Francesco; Radimerski, Thomas

    2016-01-01

    JAK inhibitors are being developed for the treatment of rheumatoid arthritis, psoriasis, myeloproliferative neoplasms and leukemias. Most of these drugs target the ATP-binding pocket and stabilize the active conformation of the JAK kinases. This type-I binding mode leads to an increase in JAK activation-loop phosphorylation, despite blockade of kinase function. Here we report that stabilizing the inactive state via type-II inhibition acts in the opposite manner, leading to a loss of activation-loop phosphorylation. We used X-ray crystallography to corroborate the binding mode and report for the first time the crystal structure of the JAK2 kinase domain in an inactive conformation. Importantly, JAK inhibitor-induced activation-loop phosphorylation requires receptor interaction, as well as intact kinase and pseudokinase domains. Hence, depending on the respective conformation stabilized by a JAK inhibitor, hyperphosphorylation of the activation-loop may or may not be elicited. PMID:22684457

  14. Characterization of inhibitor(s) of lymphocyte activation in serum from rats with adjuvant arthritis.

    PubMed

    Binderup, L; Bramm, E; Arrigoni-Martelli, E

    1978-01-01

    Serum from adjuvant arthritic rats inhibits the concanavalin A- (Con A) and lipopolysaccharide-induced stimulation of lymph node cells, leaving the basal and phytohemagglutinin-stimulated 3H-thymidine incorporation unaffected. Con A-stimulated 3H-thymidine uptake is also inhibited in rat spleen and peripheral blood lymphocytes and in dog peripheral blood lymphocytes. The intensity of the inhibitory activity in serum is positively correlated with the intensity of the secondary lesions of adjuvant arthritis. Inhibitory activity was not found in serum from rats bearing nystatin-induced inflammation. Serum fractionation studies indicated that the inhibitory activity cannot be attributed to low molecular weight alpha2-glycoproteins or to gamma-globulins and alpha2-macroglobulins, but it is present in a fraction migrating with beta-globulins. The inhibitory activity in arthritic rat serum is reduced by treatment with non-steroidal anti-inflammatory drugs, but is unaltered by D-penicillamine. It is suggested that this inhibitory activity is part of the systemic response to an immunologically mediated inflammation. PMID:151867

  15. Novel peptidomimetic inhibitors of signal transducer and activator of transcription 3 dimerization and biological activity.

    PubMed

    Turkson, James; Kim, Joon S; Zhang, Shumin; Yuan, Jing; Huang, Mei; Glenn, Matthew; Haura, Eric; Sebti, Said; Hamilton, Andrew D; Jove, Richard

    2004-03-01

    The critical role of signal transducer and activator of transcription 3 (Stat3) in the growth and survival of human tumor cells identifies it as a promising target for cancer drug discovery. We previously identified a Stat3 SH2 domain-binding phosphopeptide, PY*LKTK, and its tripeptide derivatives, PY*L and AY*L (where Y* represents phosphotyrosine), which inhibit Stat3 biochemical activity and biological function. Here, we report novel peptidomimetic compounds based on PY*L (or AY*L) with substitution of the Y-1 residue by benzyl, pyridyl, or pyrazinyl derivatives that are selective and greater than 5-fold more potent in disrupting Stat3 activity in vitro than lead tripeptides. The biological activities of these derivatives mirror that originally observed for peptides. In this context, the representative peptidomimetic ISS 610 with 4-cyanobenzoate substitution inhibits constitutive Stat3 activity in Src-transformed mouse fibroblasts and human breast and lung carcinoma cells. This effect is not evident with the non-phosphorylated counterpart, ISS 610NP, consistent with interaction of peptidomimetics with the SH2 domain of Stat3. Moreover, ISS 610 induces cell growth inhibition and apoptosis of Src-transformed fibroblasts that contain persistently active Stat3. We present the first report of a peptidomimetic approach to design of small-molecule inhibitors of Stat3 that are also among the first examples of disruptors of transcription factor dimerization with the potential for novel cancer therapy.

  16. Low-volume multiplexed proteolytic activity assay and inhibitor analysis through a pico-injector array.

    PubMed

    Ng, Ee Xien; Miller, Miles A; Jing, Tengyang; Lauffenburger, Doug A; Chen, Chia-Hung

    2015-02-21

    Secreted active proteases, from families of enzymes such as matrix metalloproteinases (MMPs) and ADAMs (a disintegrin and metalloproteinases), participate in diverse pathological processes. To simultaneously measure multiple specific protease activities, a series of parallel enzyme reactions combined with a series of inhibitor analyses for proteolytic activity matrix analysis (PrAMA) are essential but limited due to the sample quantity requirements and the complexity of performing multiple reactions. To address these issues, we developed a pico-injector array to generate 72 different reactions in picoliter-volume droplets by controlling the sequence of combinational injections, which allowed simultaneous recording of a wide range of multiple enzyme reactions and measurement of inhibitor effects using small sample volumes (~10 μL). Multiple MMP activities were simultaneously determined by 9 different substrates and 2 inhibitors using injections from a pico-injector array. Due to the advantages of inhibitor analysis, the MMP/ADAM activities of MDA-MB-231, a breast cancer cell line, were characterized with high MMP-2, MMP-3 and ADAM-10 activity. This platform could be customized for a wide range of applications that also require multiple reactions with inhibitor analysis to enhance the sensitivity by encapsulating different chemical sensors.

  17. Selective COX-2 Inhibitors: A Review of Their Structure-Activity Relationships

    PubMed Central

    Zarghi, Afshin; Arfaei, Sara

    2011-01-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are the competitive inhibitors of cyclooxygenase (COX), the enzyme which mediates the bioconversion of arachidonic acid to inflammatory prostaglandins (PGs). Their use is associated with the side effects such as gastrointestinal and renal toxicity. The therapeutic anti-inflammatory action of NSAIDs is produced by the inhibition of COX-2, while the undesired side effects arise from inhibition of COX-1 activity. Thus, it was though that more selective COX-2 inhibitors would have reduced side effects. Based upon a number of selective COX-2 inhibitors (rofecoxib, celecoxib, valdecoxibetc.) were developed as safer NSAIDs with improved gastric safety profile. However, the recent market removal of some COXIBs such as rofecoxib due to its adverse cardiovascular side effects clearly encourages the researchers to explore and evaluate alternative templates with COX-2 inhibitory activity. Recognition of new avenues for selective COX-2 inhibitors in cancer chemotherapy and neurological diseases such as Parkinson and Alzheimer’s diseases still continues to attract investigations on the development of COX-2 inhibitors. This review highlights the various structural classes of selective COX-2 inhibitors with special emphasis on their structure-activity relationships. PMID:24250402

  18. 4-Anilino-6-phenyl-quinoline inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2).

    PubMed

    Olsson, Henric; Sjö, Peter; Ersoy, Oguz; Kristoffersson, Anna; Larsson, Joakim; Nordén, Bo

    2010-08-15

    A class of inhibitors of mitogen activated protein kinase-activated kinase 2 (MK2) was discovered via high-throughput screening. This compound class demonstrates activity against the enzyme with sub-microM IC(50) values, and suppresses LPS-induced TNFalpha levels in THP-1 cells. MK2 inhibition kinetic measurements indicated mixed binding approaching non-ATP competitive inhibition.

  19. Structure-activity relationships of substituted oxyoxalamides as inhibitors of the human soluble epoxide hydrolase.

    PubMed

    Kim, In-Hae; Lee, In-Hee; Nishiwaki, Hisashi; Hammock, Bruce D; Nishi, Kosuke

    2014-02-01

    We explored both structure-activity relationships among substituted oxyoxalamides used as the primary pharmacophore of inhibitors of the human sEH and as a secondary pharmacophore to improve water solubility of inhibitors. When the oxyoxalamide function was modified with a variety of alkyls or substituted alkyls, compound 6 with a 2-adamantyl group and a benzyl group was found to be a potent sEH inhibitor, suggesting that the substituted oxyoxalamide function is a promising primary pharmacophore for the human sEH, and compound 6 can be a novel lead structure for the development of further improved oxyoxalamide or other related derivatives. In addition, introduction of substituted oxyoxalamide to inhibitors with an amide or urea primary pharmacophore produced significant improvements in inhibition potency and water solubility. In particular, the N,N,O-trimethyloxyoxalamide group in amide or urea inhibitors (26 and 31) was most effective among those tested for both inhibition and solubility. The results indicate that substituted oxyoxalamide function incorporated into amide or urea inhibitors is a useful secondary pharmacophore, and the resulting structures will be an important basis for the development of bioavailable sEH inhibitors.

  20. Designed Inhibitors of Insulin-Degrading Enzyme Regulate the Catabolism and Activity of Insulin

    PubMed Central

    Leissring, Malcolm A.; Malito, Enrico; Hedouin, Sabrine; Reinstatler, Lael; Sahara, Tomoko; Abdul-Hay, Samer O.; Choudhry, Shakeel; Maharvi, Ghulam M.; Fauq, Abdul H.; Huzarska, Malwina; May, Philip S.; Choi, Sungwoon; Logan, Todd P.; Turk, Benjamin E.; Cantley, Lewis C.; Manolopoulou, Marika; Tang, Wei-Jen; Stein, Ross L.; Cuny, Gregory D.; Selkoe, Dennis J.

    2010-01-01

    Background Insulin is a vital peptide hormone that is a central regulator of glucose homeostasis, and impairments in insulin signaling cause diabetes mellitus. In principle, it should be possible to enhance the activity of insulin by inhibiting its catabolism, which is mediated primarily by insulin-degrading enzyme (IDE), a structurally and evolutionarily distinctive zinc-metalloprotease. Despite interest in pharmacological inhibition of IDE as an attractive anti-diabetic approach dating to the 1950s, potent and selective inhibitors of IDE have not yet emerged. Methodology/Principal Findings We used a rational design approach based on analysis of combinatorial peptide mixtures and focused compound libraries to develop novel peptide hydroxamic acid inhibitors of IDE. The resulting compounds are ∼106 times more potent than existing inhibitors, non-toxic, and surprisingly selective for IDE vis-à-vis conventional zinc-metalloproteases. Crystallographic analysis of an IDE-inhibitor complex reveals a novel mode of inhibition based on stabilization of IDE's “closed,” inactive conformation. We show further that pharmacological inhibition of IDE potentiates insulin signaling by a mechanism involving reduced catabolism of internalized insulin. Conclusions/Significance The inhibitors we describe are the first to potently and selectively inhibit IDE or indeed any member of this atypical zinc-metalloprotease superfamily. The distinctive structure of IDE's active site, and the mode of action of our inhibitors, suggests that it may be possible to develop inhibitors that cross-react minimally with conventional zinc-metalloproteases. Significantly, our results reveal that insulin signaling is normally regulated by IDE activity not only extracellularly but also within cells, supporting the longstanding view that IDE inhibitors could hold therapeutic value for the treatment of diabetes. PMID:20498699

  1. Structure–activity studies with high-affinity inhibitors of pyroglutamyl-peptidase II

    PubMed Central

    2005-01-01

    Inhibitors of PPII (pyroglutamyl-peptidase II) (EC 3.4.19.6) have potential applications as investigative and therapeutic agents. The rational design of inhibitors is hindered, however, by the lack of an experimental structure for PPII. Previous studies have demonstrated that replacement of histidine in TRH (thyrotropin-releasing hormone) with asparagine produces a competitive PPII inhibitor (Ki 17.5 μM). To gain further insight into which functional groups are significant for inhibitory activity, we investigated the effects on inhibition of structural modifications to Glp-Asn-ProNH2 (pyroglutamyl-asparaginyl-prolineamide). Synthesis and kinetic analysis of a diverse series of carboxamide and C-terminally extended Glp-Asn-ProNH2 analogues were undertaken. Extensive quantitative structure–activity relationships were generated, which indicated that key functionalities in the basic molecular structure of the inhibitors combine in a unique way to cause PPII inhibition. Data from kinetic and molecular modelling studies suggest that hydrogen bonding between the asparagine side chain and PPII may provide a basis for the inhibitory properties of the asparagine-containing peptides. Prolineamide appeared to be important for interaction with the S2′ subsite, but some modifications were tolerated. Extension of Glp-Asn-ProNH2 with hydrophobic amino acids at the C-terminus led to a novel set of PPII inhibitors active in vitro at nanomolar concentrations. Such inhibitors were shown to enhance recovery of TRH released from rat brain slices. Glp-Asn-Pro-Tyr-Trp-Trp-7-amido-4-methylcoumarin displayed a Ki of 1 nM, making it the most potent competitive PPII inhibitor described to date. PPII inhibitors with this level of potency should find application in exploring the biological functions of TRH and PPII, and potentially provide a basis for development of novel therapeutics. PMID:15799721

  2. Designed Inhibitors of Insulin-Degrading Enzyme Regulate the Catabolism and Activity of Insulin

    SciTech Connect

    Leissring, Malcolm A.; Malito, Enrico; Hedouin, Sabrine; Reinstatler, Lael; Sahara, Tomoko; Abdul-Hay, Samer O.; Choudhry, Shakeel; Maharvi, Ghulam M.; Fauq, Abdul H.; Huzarska, Malwina; May, Philip S.; Choi, Sungwoon; Logan, Todd P.; Turk, Benjamin E.; Cantley, Lewis C.; Manolopoulou, Marika; Tang, Wei-Jen; Stein, Ross L.; Cuny, Gregory D.; Selkoe, Dennis J.

    2010-09-20

    Insulin is a vital peptide hormone that is a central regulator of glucose homeostasis, and impairments in insulin signaling cause diabetes mellitus. In principle, it should be possible to enhance the activity of insulin by inhibiting its catabolism, which is mediated primarily by insulin-degrading enzyme (IDE), a structurally and evolutionarily distinctive zinc-metalloprotease. Despite interest in pharmacological inhibition of IDE as an attractive anti-diabetic approach dating to the 1950s, potent and selective inhibitors of IDE have not yet emerged. We used a rational design approach based on analysis of combinatorial peptide mixtures and focused compound libraries to develop novel peptide hydroxamic acid inhibitors of IDE. The resulting compounds are {approx} 10{sup 6} times more potent than existing inhibitors, non-toxic, and surprisingly selective for IDE vis-a-vis conventional zinc-metalloproteases. Crystallographic analysis of an IDE-inhibitor complex reveals a novel mode of inhibition based on stabilization of IDE's 'closed,' inactive conformation. We show further that pharmacological inhibition of IDE potentiates insulin signaling by a mechanism involving reduced catabolism of internalized insulin. Conclusions/Significance: The inhibitors we describe are the first to potently and selectively inhibit IDE or indeed any member of this atypical zinc-metalloprotease superfamily. The distinctive structure of IDE's active site, and the mode of action of our inhibitors, suggests that it may be possible to develop inhibitors that cross-react minimally with conventional zinc-metalloproteases. Significantly, our results reveal that insulin signaling is normally regulated by IDE activity not only extracellularly but also within cells, supporting the longstanding view that IDE inhibitors could hold therapeutic value for the treatment of diabetes.

  3. Inhibitory effects of harpagoside on TNF-α-induced pro-inflammatory adipokine expression through PPAR-γ activation in 3T3-L1 adipocytes.

    PubMed

    Kim, Tae Kon; Park, Kyoung Sik

    2015-12-01

    Obesity is closely associated with increased production of pro-inflammatory adipokines, including interleukin (IL)-6, plasminogen activator inhibitor (PAI)-1, and adipose-tissue-derived monocyte chemoattractant protein (MCP)-1, which contribute to chronic and low-grade inflammation in adipose tissue. Harpagoside, a major iridoid glycoside present in devil's claw, has been reported to show anti-inflammatory activities by suppression of lipopolysaccharide (LPS)-induced production of inflammatory cytokines in murine macrophages. The present study is aimed to investigate the effects of harpagoside on both tumor necrosis factor (TNF)-α-induced inflammatory adipokine expression and its underlying signaling pathways in differentiated 3T3-L1 cells. Harpagoside significantly inhibited TNF-α-induced mRNA synthesis and protein production of the atherogenic adipokines including IL-6, PAI-1, and MCP-1. Further investigation of the molecular mechanism revealed that pretreatment with harpagoside activated peroxisome proliferator-activated receptor (PPAR)-γ. These findings suggest that the clinical application of medicinal plants which contain harpagoside may lead to a partial prevention of obesity-induced atherosclerosis by attenuating inflammatory responses. PMID:26049170

  4. Discovery of orally active pyrrolopyridine- and aminopyridine-based Met kinase inhibitors

    SciTech Connect

    Cai, Zhen-Wei; Wei, Donna; Schroeder, Gretchen M.; Cornelius, Lyndon A.M.; Kim, Kyoung; Chen, Xiao-Tao; Schmidt, Robert J.; Williams, David K.; Tokarski, John S.; An, Yongmi; Sack, John S.; Manne, Veeraswamy; Kamath, Amrita; Zhang, Yueping; Marathe, Punit; Hunt, John T.; Lombardo, Louis J.; Fargnoli, Joseph; Borzilleri, Robert M.

    2008-09-10

    A series of acylurea analogs derived from pyrrolopyridine and aminopyridine scaffolds were identified as potent inhibitors of Met kinase activity. The SAR at various positions of the two kinase scaffolds was investigated. These studies led to the discovery of compounds 3b and 20b, which demonstrated favorable pharmacokinetic properties in mice and significant antitumor activity in a human gastric carcinoma xenograft model.

  5. Acylprolinamides: a new class of peptide deformylase inhibitors with in vivo antibacterial activity.

    PubMed

    Axten, Jeffrey M; Medina, Jesús R; Blackledge, Charles W; Duquenne, Céline; Grant, Seth W; Bobko, Mark A; Peng, Tony; Miller, William H; Pinckney, Theresa; Gallagher, Timothy F; Kulkarni, Swarupa; Lewandowski, Thomas; Van Aller, Glenn S; Zonis, Rimma; Ward, Paris; Campobasso, Nino

    2012-06-15

    A new class of PDF inhibitor with potent, broad spectrum antibacterial activity is described. Optimization of blood stability and potency provided compounds with improved pharmacokinetics that were suitable for in vivo experiments. Compound 5c, which has robust antibacterial activity, demonstrated efficacy in two respiratory tract infection models.

  6. Ku80 as a novel receptor for thymosin beta4 that mediates its intracellular activity different from G-actin sequestering.

    PubMed

    Bednarek, Radoslaw; Boncela, Joanna; Smolarczyk, Katarzyna; Cierniewska-Cieslak, Aleksandra; Wyroba, Elzbieta; Cierniewski, Czeslaw S

    2008-01-18

    Our data demonstrate that increased intracellular expression of thymosin beta4(Tbeta4) is necessary and sufficient to induce plasminogen activator inhibitor type 1 (PAI-1) gene expression in endothelial cells. To describe the mechanism of this effect, we produced Tbeta4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of Tbeta4((AcSDKPT/4A)), Tbeta4((KLKKTET/7A)), and Tbeta4((K16A)) mutants fused with green fluorescent protein did not differ significantly from those of wild-type Tbeta4. Overexpression of Tbeta4, Tbeta4((AcSDKPT/4A)), and Tbeta4((K16A)) affected intracellular formation of actin filaments. As expected, Tbeta4((K16A)) uptake by nuclei was impaired. On the other hand, overexpression of Tbeta4((KLKKTET/7A)) resulted in developing the actin filament network typical of adhering cells, indicating that the mutant lacked the actin binding site. The mechanism by which intracellular Tbeta4 induced the PAI-1 gene did not depend upon the N-terminal tetrapeptide AcSDKP and depended only partially on its ability to bind G-actin or enter the nucleus. Both Tbeta4 and Tbeta4((AcSDKPT/4A)) induced the PAI-1 gene to the same extent, whereas mutants Tbeta4((KLKKTET/7A)) and Tbeta4((K16A)) retained about 60% of the original activity. By proteomic analysis, the Ku80 subunit of ATP-dependent DNA helicase II was found to be associated with Tbeta4. Ku80 and Tbeta4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and Tbeta4 showed that the C-terminal arm domain of Ku80 is directly involved in this interaction. Furthermore, down-regulation of Ku80 by specific short interference RNA resulted in dramatic reduction in PAI-1 expression at the level of both mRNA and protein synthesis. These data suggest that Ku80 functions as a novel receptor for Tbeta4 and mediates its intracellular activity.

  7. A New Class of Rhomboid Protease Inhibitors Discovered by Activity-Based Fluorescence Polarization

    PubMed Central

    Wolf, Eliane V.; Zeißler, Annett; Vosyka, Oliver; Zeiler, Evelyn; Sieber, Stephan; Verhelst, Steven H. L.

    2013-01-01

    Rhomboids are intramembrane serine proteases that play diverse biological roles, including some that are of potential therapeutical relevance. Up to date, rhomboid inhibitor assays are based on protein substrate cleavage. Although rhomboids have an overlapping substrate specificity, substrates cannot be used universally. To overcome the need for substrates, we developed a screening assay using fluorescence polarization activity-based protein profiling (FluoPol ABPP) that is compatible with membrane proteases. With FluoPol ABPP, we identified new inhibitors for the E. coli rhomboid GlpG. Among these was a structural class that has not yet been reported as rhomboid inhibitors: β-lactones. They form covalent and irreversible complexes with the active site serine of GlpG. The presence of alkyne handles on the β-lactones also allowed activity-based labeling. Overall, these molecules represent a new scaffold for future inhibitor and activity-based probe development, whereas the assay will allow inhibitor screening of ill-characterized membrane proteases. PMID:23991088

  8. Effect of reactive site loop elongation on the inhibitory activity of C1-inhibitor.

    PubMed

    Bos, Ineke G A; Lubbers, Yvonne T P; Eldering, Eric; Abrahams, Jan Pieter; Hack, C Erik

    2004-06-01

    The serine protease inhibitor C1-Inhibitor (C1-Inh) inhibits several complement- and contact-system proteases, which play an important role in inflammation. C1-Inh has a short reactive site loop (RSL) compared to other serpins. RSL length determines the inhibitory activity of serpins. We investigated the effect of RSL elongation on inhibitory activity of C1-Inh by insertion of one or two alanine residues in the RSL. One of five mutants had an increased association rate with kallikrein, but was nevertheless a poor inhibitor because of a simultaneous high stoichiometry of inhibition (>10). The association rate of the other variants was lower than that of wild-type C1-Inh. These data suggest that the relatively weak inhibitory activity of C1-Inh is not the result of its short RSL. The short RSL of C1-Inh has, surprisingly, the optimal length for inhibition.

  9. Highly improved antiparasitic activity after introduction of an N-benzylimidazole moiety on protein farnesyltransferase inhibitors.

    PubMed

    Bosc, Damien; Mouray, Elisabeth; Cojean, Sandrine; Franco, Caio Haddad; Loiseau, Philippe M; Freitas-Junior, Lucio H; Moraes, Carolina Borsoi; Grellier, Philippe; Dubois, Joëlle

    2016-02-15

    In our search for new protein farnesyltransferase inhibitors with improved antiparasitic activities, we modified our previously developed 3-arylthiophene series of inhibitors by replacing the thioisopropyl group by different substituted imidazolylmethanamino moieties. Twenty four new derivatives were synthesized and evaluated against human and parasite farnesyltransferases, and their anti-parasitic activity was determined against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani. Introduction of a N-p-substituted-benzylimidazole led to significantly increase the inhibition of parasite proliferation in the submicromolar range. The structure of the best inhibitors was parasite dependent. Three compounds possess IC50 values at the same range as the reference miltefosine against L. donovani proliferation and other new derivatives display high level of anti-trypanosomal activity against T. cruzi, higher or in the same order of magnitude as the reference compounds benznidazole and nifurtimox. PMID:26774924

  10. PHOTOREGULATION OF BIOLOGICAL ACTIVITY BY PHOTOCROMIC REAGENTS, II. INHIBITORS OF ACETYLCHOLINESTERASE*†

    PubMed Central

    Bieth, Joseph; Vratsanos, Spyros M.; Wassermann, Norbert; Erlanger, Bernard F.

    1969-01-01

    The enzymic activity of acetylcholinesterase can be photoregulated through the mediation of photochromic inhibitors of the enzyme. N-p-phenylazophenyl-N-phenylcarbamyl fluoride, an irreversible inhibitor of acetylcholinesterase, exists as two geometric isomers which are interconvertible through the action of light. The cis isomer, which predominates after exposure to light of 320 nm, is more active than the trans isomer, which results from exposure to light of 420 nm. It was possible, therefore, to use light energy to regulate the inactivation of the enzyme. Similarly, levels of acetylcholinesterase activity could be photo-regulated in a completely reversible manner by means of the photochromic reversible inhibitor p-phenylazophenyltrimethylammonium chloride. These experiments can serve as models for similar phenomena observed in nature, particularly in photoperiodic rhythms of higher animals. Images PMID:5264140

  11. Self-Reported Sitting Time, Physical Activity and Fibrinolytic and Other Novel Cardio-Metabolic Biomarkers in Active Swedish Seniors

    PubMed Central

    Howard, Bethany J.; Hurtig-Wennlöf, Anita; Olsson, Lovisa A.; Nilsson, Torbjörn K.; Dunstan, David W.; Wennberg, Patrik

    2016-01-01

    Background Too much sitting is linked with an increased risk of cardiovascular disease and mortality. The mediating mechanisms for these associations are largely unknown, however dysregulated fibrinolysis have emerged as a possible contributor. Objective We examined the associations of self-reported overall sitting time and physical activity with fibrinolytic and other novel cardio-metabolic biomarkers in older adults. Materials and Methods Data was analysed for 364 participants (74±7 yrs) of the Active Seniors group (retired, living independently in their own homes). Linear regression analyses examined associations of categories of categories of sitting time (≤3, 3–6, >6 hrs/day) and overall physical activity (Low, Moderate and High) with biomarkers in serum or plasma, adjusting for age, gender and smoking (with further adjustment for either overall physical activity or sitting time and BMI in secondary analyses). Results Compared to sitting ≤ 3 hrs/day, sitting >6 hrs/day was associated with higher tissue plasminogen activator (tPA) and tissue plasminogen activator/plasminogen activator inhibitor-1 complex (tPA-PAI-1 complex). These associations were not independent of overall physical activity or BMI. Compared to those in the high physical activity, low physical activity was associated with a higher BMI, high-sensitivity C-reactive protein (hs-CRP) and tPA-PAI-1 complex levels. Only the associations of BMI and hs-CRP were independent of sitting time. Conclusions These findings provide preliminary cross-sectional evidence for the relationships of sitting time with fibrinolytic markers in older adults. They also reinforce the importance of regular physical activity for cardio-metabolic health. PMID:27658041

  12. Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening

    PubMed Central

    Chormova, Dimitra; Franková, Lenka; Defries, Andrew; Cutler, Sean R.; Fry, Stephen C.

    2015-01-01

    Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes’ biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme–cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors – especially compounds that generate singlet oxygen (1O2) e.g., riboflavin (IC50 29 μM), retinoic acid, eosin (IC50 27 μM) and erythrosin (IC50 36 μM). The riboflavin effect was light-dependent, supporting 1O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (−)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1 μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 μM mycophenolic acid and (−)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads. PMID:26093490

  13. Discovery of small molecule inhibitors of xyloglucan endotransglucosylase (XET) activity by high-throughput screening.

    PubMed

    Chormova, Dimitra; Franková, Lenka; Defries, Andrew; Cutler, Sean R; Fry, Stephen C

    2015-09-01

    Small molecules (xenobiotics) that inhibit cell-wall-localised enzymes are valuable for elucidating the enzymes' biological roles. We applied a high-throughput fluorescent dot-blot screen to search for inhibitors of Petroselinum xyloglucan endotransglucosylase (XET) activity in vitro. Of 4216 xenobiotics tested, with cellulose-bound xyloglucan as donor-substrate, 18 inhibited XET activity and 18 promoted it (especially anthraquinones and flavonoids). No compounds promoted XET in quantitative assays with (cellulose-free) soluble xyloglucan as substrate, suggesting that promotion was dependent on enzyme-cellulose interactions. With cellulose-free xyloglucan as substrate, we found 22 XET-inhibitors - especially compounds that generate singlet oxygen ((1)O2) e.g., riboflavin (IC50 29 μM), retinoic acid, eosin (IC50 27 μM) and erythrosin (IC50 36 μM). The riboflavin effect was light-dependent, supporting (1)O2 involvement. Other inhibitors included tannins, sulphydryl reagents and triphenylmethanes. Some inhibitors (vulpinic acid and brilliant blue G) were relatively specific to XET, affecting only two or three, respectively, of nine other wall-enzyme activities tested; others [e.g. (-)-epigallocatechin gallate and riboflavin] were non-specific. In vivo, out of eight XET-inhibitors bioassayed, erythrosin (1 μM) inhibited cell expansion in Rosa and Zea cell-suspension cultures, and 40 μM mycophenolic acid and (-)-epigallocatechin gallate inhibited Zea culture growth. Our work showcases a general high-throughput strategy for discovering wall-enzyme inhibitors, some being plant growth inhibitors potentially valuable as physiological tools or herbicide leads. PMID:26093490

  14. The potent Cdc7-Dbf4 (DDK) kinase inhibitor XL413 has limited activity in many cancer cell lines and discovery of potential new DDK inhibitor scaffolds.

    PubMed

    Sasi, Nanda Kumar; Tiwari, Kanchan; Soon, Fen-Fen; Bonte, Dorine; Wang, Tong; Melcher, Karsten; Xu, H Eric; Weinreich, Michael

    2014-01-01

    Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase) is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC50 values against the purified kinase. Although XL413 is highly selective for DDK, its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491, whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly, their activities in cell lines were highly divergent. Unlike PHA-767491, XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines, this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors, we also tested the cross-reactivity of ∼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA). We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491, including Chk1 and PKR kinase inhibitors, but had divergent chemical scaffolds from known DDK inhibitors. Taken together, these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific, biologically active DDK inhibitors for use as chemotherapeutic agents.

  15. The Potent Cdc7-Dbf4 (DDK) Kinase Inhibitor XL413 Has Limited Activity in Many Cancer Cell Lines and Discovery of Potential New DDK Inhibitor Scaffolds

    PubMed Central

    Sasi, Nanda Kumar; Tiwari, Kanchan; Soon, Fen-Fen; Bonte, Dorine; Wang, Tong; Melcher, Karsten; Xu, H. Eric; Weinreich, Michael

    2014-01-01

    Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase) is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC50 values against the purified kinase. Although XL413 is highly selective for DDK, its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491, whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly, their activities in cell lines were highly divergent. Unlike PHA-767491, XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines, this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors, we also tested the cross-reactivity of ∼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA). We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491, including Chk1 and PKR kinase inhibitors, but had divergent chemical scaffolds from known DDK inhibitors. Taken together, these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific, biologically active DDK inhibitors for use as chemotherapeutic agents. PMID:25412417

  16. Synthesis of non-competitive inhibitors of sphingomyelinases with significant activity.

    PubMed

    Yokomatsu, Tsutomu; Murano, Tetsuo; Akiyama, Takeshi; Koizumi, Junichi; Shibuya, Shiroshi; Tsuji, Yoshiaki; Soeda, Shinji; Shimeno, Hiroshi

    2003-01-20

    A series of short-chain analogues of N-palmitoylsphingosine-1-phosphate, modified by replacement of the phosphate and the long alkenyl side chain with hydrolytically stable difluoromethylene phosphonate and phenyl, respectively, were prepared to study the structure-activity relationship for inhibition of sphingomyelinase. The study revealed that inhibition is highly dependent upon the stereochemistry of the asymmetric centers of the acylamino moiety, and resulted in identification of a non-competitive inhibitor with the same level of inhibitory activity of schyphostatin, the most potent of the few known small molecular inhibitors of sphingomyelinase.

  17. Synthesis and structure-activity relationships of potent 4-fluoro-2-cyanopyrrolidine dipeptidyl peptidase IV inhibitors.

    PubMed

    Fukushima, Hiroshi; Hiratate, Akira; Takahashi, Masato; Mikami, Ayako; Saito-Hori, Masako; Munetomo, Eiji; Kitano, Kiyokazu; Chonan, Sumi; Saito, Hidetaka; Suzuki, Akio; Takaoka, Yuji; Yamamoto, Koji

    2008-04-01

    Dipeptidyl peptidase IV (DPP-IV) inhibitors are promising antidiabetic drugs, and several drugs are in the developmental stage. We previously reported that the introduction of fluorine to the 4-position of 2-cyanopyrrolidine enhanced the DPP-IV inhibitory effect. In the present report, we examined the structure-activity relationship (SAR) of 2-cyano-4-fluoropyrrolidine with N-substituted glycine at the 1-position. We report the identification of a potent and stable DPP-IV inhibitor (TS-021) with a long-term persistent plasma drug concentration and a potent antihyperglycemic activity.

  18. New direct inhibitors of InhA with antimycobacterial activity based on a tetrahydropyran scaffold.

    PubMed

    Pajk, Stane; Živec, Matej; Šink, Roman; Sosič, Izidor; Neu, Margarete; Chung, Chun-wa; Martínez-Hoyos, María; Pérez-Herrán, Esther; Álvarez-Gómez, Daniel; Álvarez-Ruíz, Emilio; Mendoza-Losana, Alfonso; Castro-Pichel, Julia; Barros, David; Ballell-Pages, Lluís; Young, Robert J; Convery, Maire A; Encinas, Lourdes; Gobec, Stanislav

    2016-04-13

    Tetrahydropyran derivative 1 was discovered in a high-throughput screening campaign to find new inhibitors of mycobacterial InhA. Following initial in-vitro profiling, a structure-activity relationship study was initiated and a focused library of analogs was synthesized and evaluated. This yielded compound 42 with improved antimycobacterial activity and low cytotoxicity. Additionally, the crystal structure of InhA in complex with inhibitor 1 was resolved, to reveal the binding mode and provide hints for further optimization. PMID:26900657

  19. A Trypsin Inhibitor from Clitoria fairchildiana Cotyledons is Active Against Digestive Enzymes of Aedes aegypti Larvae.

    PubMed

    de Oliveira, Lucilene O; Fernandes, Kátia V S; Pádua, Dayanni de Souza; Carvalho, André de O; Lemos, Francisco J A; Gomes, Valdirene M; Oliveira, Antônia E A; Ferreira, André T da Silva; Perales, Jonas

    2015-01-01

    Aedes aegypti, the principal mosquito vector of yellow fever, dengue fever and chikungunya fever virus-transmitted diseases, is an insect closely associated with humans and their housing habitats. As there is no commercially available vaccine, prevention is the most suggested form of avoiding disease spreading and a number of studies are being developed in order to give support to vector control operations. The present study reports on the identification of a trypsin inhibitor isolated from cotyledons of the Clitoria fairchildiana amazonic tree seeds, which was able to reduce by 87.93 % the activity of digestive enzymes of fourth instar A. aegypti larva. A partial amino acid sequence showed strong similarity with sequences from several trypsin inhibitors already reported in the literature. The 13,000 Da isolated inhibitor was seen to be active solely against trypsin-like enzymes, neither acting on papain, α-amylase nor on other serine proteases, such as elastase, chymotrypsin or subtilisin. At least six from seven active digestive proteases from A. aegypti larvae, visualized by zymography, were severely affected soon after exposed to the inhibitor. The strong and specific action of the isolated inhibitor against trypsin digestive enzymes of this insect vector led us to believe that this protein may be a good candidate for a prospective alternative biocontrol method. PMID:26156641

  20. Polycomb repressive complex 2 structure with inhibitor reveals a mechanism of activation and drug resistance

    PubMed Central

    Brooun, Alexei; Gajiwala, Ketan S.; Deng, Ya-Li; Liu, Wei; Bolaños, Ben; Bingham, Patrick; He, You-Ai; Diehl, Wade; Grable, Nicole; Kung, Pei-Pei; Sutton, Scott; Maegley, Karen A.; Yu, Xiu; Stewart, Al E.

    2016-01-01

    Polycomb repressive complex 2 (PRC2) mediates gene silencing through chromatin reorganization by methylation of histone H3 lysine 27 (H3K27). Overexpression of the complex and point mutations in the individual subunits of PRC2 have been shown to contribute to tumorigenesis. Several inhibitors of the PRC2 activity have shown efficacy in EZH2-mutated lymphomas and are currently in clinical development, although the molecular basis of inhibitor recognition remains unknown. Here we report the crystal structures of the inhibitor-bound wild-type and Y641N PRC2. The structures illuminate an important role played by a stretch of 17 residues in the N-terminal region of EZH2, we call the activation loop, in the stimulation of the enzyme activity, inhibitor recognition and the potential development of the mutation-mediated drug resistance. The work presented here provides new avenues for the design and development of next-generation PRC2 inhibitors through establishment of a structure-based drug design platform. PMID:27122193

  1. A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase

    PubMed Central

    Morgan, Hugh P.; Walsh, Martin J.; Blackburn, Elizabeth A.; Wear, Martin A.; Boxer, Matthew B.; Shen, Min; Mcnae, Iain W.; Nowicki, Matthew W.; Michels, Paul A. M.; Auld, Douglas S.; Fothergill-Gilmore, Linda A.; Walkinshaw, Malcolm D.

    2012-01-01

    SYNOPSIS Pyruvate kinase (PYK) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit Leishmania mexicana PYK (LmPYK) activity in a time- (and dose-) dependent manner; a characteristic of irreversible inhibition. The crystal structure of 4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid (DBS) complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors. PMID:22906073

  2. p38 Mitogen-activated protein kinase inhibitors: a review on pharmacophore mapping and QSAR studies.

    PubMed

    Gangwal, Rahul P; Bhadauriya, Anuseema; Damre, Mangesh V; Dhoke, Gaurao V; Sangamwar, Abhay T

    2013-01-01

    p38 mitogen-activated protein (MAP) kinases are the serine/threonine protein kinases, which play a vital role in cellular responses to external stress signals. p38 MAP kinase inhibitors have shown anti-inflammatory effects in the preclinical disease models, primarily through inhibition of the expression of inflammatory mediators. A number of structurally diverse p38 MAP kinase inhibitors have been developed as potential anti-inflammatory agents. Most of the inhibitors have failed in the clinical trials either due to poor pharmacokinetic profile or selectivity issue, which makes p38 MAP kinase a promising target for molecular modelling studies. Several quantitative structure activity relationships (QSAR) and pharmacophore models have been developed to identify the structural requirements essential for p38 MAP kinase inhibitory activity. In this review, we provide an overview of the presently known p38 MAP kinase inhibitors and how QSAR analyses among series of compounds have led to the development of molecular models and pharmacophores, allowing the design of novel inhibitors.

  3. Type 1 plaminogen activator inhibitor gene: Functional analysis and glucocorticoid regulation of its promoter

    SciTech Connect

    Van Zonneveld, A.J.; Curriden, S.A.; Loskutoff, D.J. )

    1988-08-01

    Plasminogen activator inhibitor type 1 is an important component of the fibrinolytic system and its biosynthesis is subject to complex regulation. To study this regulation at the level of transcription, the authors have identified and sequenced the promoter of the human plasminogen activator inhibitor type 1 gene. Nuclease protection experiments were performed by using endothelial cell mRNA and the transcription initiation (cap) site was established. Sequence analysis of the 5{prime} flanking region of the gene revealed a perfect TATA box at position {minus}28 to position {minus}23, the conserved distance from the cap site. Comparative functional studies with the firefly luciferase gene as a reporter gene showed that fragments derived from this 5{prime} flanking region exhibited high promoter activity when transfected into bovine aortic endothelial cells and mouse Ltk{sup {minus}} fibroblasts but were inactive when introduced into HeLa cells. These studies indicate that the fragments contain the plasminogen activator inhibitor type 1 promoter and that it is expressed in a tissue-specific manner. Although the fragments were also silent in rat FTO2B hepatoma cells, their promoter activity could be induced up to 40-fold with the synthetic glucocorticoid dexamethasone. Promoter deletion mapping experiments and studies involving the fusion of promoter fragments to a heterologous gene indicated that dexamethasone induction is mediated by a glucocorticoid responsive element with enhancer-like properties located within the region between nucleotides {minus}305 and +75 of the plasminogen activator inhibitor type 1 gene.

  4. Electron transport chain inhibitors induce microglia activation through enhancing mitochondrial reactive oxygen species production.

    PubMed

    Ye, Junli; Jiang, Zhongxin; Chen, Xuehong; Liu, Mengyang; Li, Jing; Liu, Na

    2016-01-15

    Reactive oxygen species (ROS) are believed to be mediators of excessive microglial activation, yet the resources and mechanism are not fully understood. Here we stimulated murine microglial BV-2 cells and primary microglial cells with different inhibitors of electron transport chain (ETC), rotenone, thenoyltrifluoroacetone (TTFA), antimycin A, and NaN3 to induce mitochondrial ROS production and we observed the role of mitochondrial ROS in microglial activation. Our results showed that ETC inhibitors resulted in significant changes in cell viability, microglial morphology, cell cycle arrest and mitochondrial ROS production in a dose-dependent manner in both primary cultural microglia and BV-2 cell lines. Moreover, ETC inhibitors, especially rotenone and antimycin A stimulated secretion of interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 12 (IL-12) and tumor necrosis factor α (TNF-α) by microglia with marked activation of mitogen-activated proteinkinases (MAPKs) and nuclear factor κB (NF-κB), which could be blocked by specific inhibitors of MAPK and NF-κB and mitochondrial antioxidants, Mito-TEMPO. Taken together, our results demonstrated that inhibition of mitochondrial respiratory chain in microglia led to production of mitochondrial ROS and therefore may activate MAPK/NF-кB dependent inflammatory cytokines release in microglia, which indicated that mitochondrial-derived ROS were contributed to microglial activation.

  5. Identification of Selective and Potent Inhibitors of Fibroblast Activation Protein and Prolyl Oligopeptidase

    PubMed Central

    Poplawski, Sarah E.; Lai, Jack H.; Li, Youhua; Jin, Zhiping; Liu, Yuxin; Wu, Wengen; Wu, Yong; Zhou, Yuhong; Sudmeier, James L.; Sanford, David G.; Bachovchin, William W.

    2014-01-01

    Fibroblast activation protein (FAP) is a serine protease selectively expressed on reactive stromal fibroblasts of epithelial carcinomas. It is widely believed to play a role in tumor invasion and metastasis and therefore to represent a potential new drug target for cancer. Investigation into its biological function, however, has been hampered by the current unavailability of selective inhibitors. The challenge has been in identifying inhibitors that are selective for FAP over both the dipeptidyl peptidases (DPPs), with which it shares exopeptidase specificity, and prolyl oligopeptidase (PREP), with which it shares endopeptidase specificity. Here, we report the first potent FAP inhibitor with selectivity over both the DPPs and PREP, N-(pyridine-4-carbonyl)-d-Ala-boroPro (ARI-3099, 6). We also report a similarly potent and selective PREP inhibitor, N-(pyridine-3-carbonyl)-Val-boroPro (ARI-3531, 22). Both are boronic acid based inhibitors, demonstrating that high selectivity can be achieved using this electrophile. The inhibitors are stable, easy to synthesize, and should prove to be useful in helping to elucidate the biological functions of these two unique and interesting enzymes, as well as their potential as drug targets. PMID:23594271

  6. In silico, in vitro and cellular analysis with a kinome-wide inhibitor panel correlates cellular LRRK2 dephosphorylation to inhibitor activity on LRRK2.

    PubMed

    Vancraenenbroeck, Renée; De Raeymaecker, Joren; Lobbestael, Evy; Gao, Fangye; De Maeyer, Marc; Voet, Arnout; Baekelandt, Veerle; Taymans, Jean-Marc

    2014-01-01

    Leucine-rich repeat kinase 2 (LRRK2) is a complex, multidomain protein which is considered a valuable target for potential disease-modifying therapeutic strategies for Parkinson's disease (PD). In mammalian cells and brain, LRRK2 is phosphorylated and treatment of cells with inhibitors of LRRK2 kinase activity can induce LRRK2 dephosphorylation at a cluster of serines including Ser910/935/955/973. It has been suggested that phosphorylation levels at these sites reflect LRRK2 kinase activity, however kinase-dead variants of LRRK2 or kinase activating variants do not display altered Ser935 phosphorylation levels compared to wild type. Furthermore, Ser910/935/955/973 are not autophosphorylation sites, therefore, it is unclear if inhibitor induced dephosphorylation depends on the activity of compounds on LRRK2 or on yet to be identified upstream kinases. Here we used a panel of 160 ATP competitive and cell permeable kinase inhibitors directed against all branches of the kinome and tested their activity on LRRK2 in vitro using a peptide-substrate-based kinase assay. In neuronal SH-SY5Y cells overexpressing LRRK2 we used compound-induced dephosphorylation of Ser935 as readout. In silico docking of selected compounds was performed using a modeled LRRK2 kinase structure. Receiver operating char