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

2011 ◽  
Vol 301 (6) ◽  
pp. L908-L916 ◽  
Author(s):  
Shintaro Miyamoto ◽  
Noboru Hattori ◽  
Tadashi Senoo ◽  
Yojiro Onari ◽  
Hiroshi Iwamoto ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Plasminogen Activator Inhibitor ◽  
Healthy Controls ◽  
Wild Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Asthma Model ◽  
Interfering Rna ◽  
Deficient Mice ◽  
Activator Inhibitor ◽  
Pai 1

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.

Enhanced Fibrinolytic Potential in Mice with Combined Homozygous Deficiency of α2-antiplasmin and PAI-1

2001 ◽  
Vol 86 (08) ◽  
pp. 640-646 ◽  
Author(s):  
D. Collen ◽  
H. R. Lijnen ◽  
M. Dewerchin
Keyword(s):  
Plasminogen Activator Inhibitor ◽  
Fibrin Deposition ◽  
Wild Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Fibrinolytic Potential ◽  
Fibrinolytic Capacity ◽  
Deficient Mice ◽  
Activator Inhibitor ◽  
Pai 1 ◽  
Combined Deficiency

Summaryα2-antiplasmin (α2-AP) and plasminogen activator inhibitor-1 (PAI-1) are the main physiological inhibitors of the plasminogen/ plasmin system in mammalian plasma. In the present study, the relative importance of both inhibitors was evaluated with the use of mice with single or combined deficiency of α2-AP and PAI-1 in the same genetic background. Mice with combined deficiency (α2-AP–/–:PAI-1–/–) are viable, develop normally and are fertile. After amputation of the tail, bleeding times are prolonged (>15 min) in α2-AP–/–:PAI-1–/– mice, as compared to double wild-type or single deficient mice (4.6 to 10 min). Spontaneous lysis after 4 h of intravenously injected 125I-fibrin labeled plasma clots is significantly higher in mice with α2-AP deficiency both in the PAI-1+/+ background (89 ± 2% versus 42 ± 3%; p = 0.002) and in the PAI-1–/– background (83 ± 4% versus 53 ± 5%; p = 0.002). PAI-1 deletion in the α2-AP+/+ or α2-AP–/– background, however, has no significant effect (p = 0.13 or 0.18, respectively). Four hours after endotoxin injection, fibrin deposition in the kidneys is not significantly affected by PAI-1 deletion in mice with α2-AP+/+ or α2-AP–/– background (p = 0.07 and 0.19, respectively). In contrast, α2-AP deletion causes significantly reduced fibrin deposition in the PAI-1+/+ background (p = 0.01). Endotoxin injection causes a dramatic increase in PAI-1 antigen levels in kidney extracts of PAI-1+/+ animals, without effect on α2-AP levels.Taken together, these data indicate that the higher endogenous fibrinolytic capacity observed in mice with combined deficiency is mainly due to the lack of α2-AP and suggest a less important role for PAI-1.

Vascular release of plasminogen activator inhibitor-1 impairs fibrinolysis during acute arterial thrombosis in mice

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 153-160
Author(s):  
Tomihisa Kawasaki ◽  
Mieke Dewerchin ◽  
Henri R. Lijnen ◽  
Jos Vermylen ◽  
Marc F. Hoylaerts
Keyword(s):  
Carotid Artery ◽  
Plasminogen Activator ◽  
Thrombus Formation ◽  
Blood Platelets ◽  
Plasma Concentrations ◽  
Plasminogen Activator Inhibitor ◽  
Plasminogen Activator Inhibitor 1 ◽  
Deficient Mice ◽  
Activator Inhibitor ◽  
Pai 1

The role of plasminogen activator inhibitor-1 (PAI-1) in the plasma, blood platelets, and vessel wall during acute arterial thrombus formation was investigated in gene-deficient mice. Photochemically induced thrombosis in the carotid artery was analyzed via transillumination. In comparison to thrombosis in C57BL/6J wild-type (wt) mice (113 ± 19 × 106 arbitrary light units [AU] n = 15, mean ± SEM), thrombosis in PAI-1−/− mice (40 ± 10 × 106 AU, n = 13) was inhibited (P < .01), indicating that PAI-1 controls fibrinolysis during thrombus formation. Systemic administration of murine PAI-1 into PAI-1−/− mice led to a full recovery of thrombotic response. Occurrence of fibrinolytic activity was confirmed in 2-antiplasmin (2-AP)–deficient mice. The sizes of thrombi developing in wt mice, in 2-AP+/− and 2-AP−/− mice were 102 ± 35, 65 ± 8.1, and 13 ± 6.1 × 106 AU, respectively (n = 6 each) (P < .05), compatible with functional plasmin inhibition by 2-AP. In contrast, thrombi in wt mice, t-PA−/− and u-PA−/−mice were comparable, substantiating efficient inhibition of fibrinolysis by the combined PAI-1/2-AP action. Platelet depletion and reconstitution confirmed a normal thrombotic response in wt mice, reconstituted with PAI-1−/− platelets, but weak thrombosis in PAI-1−/− mice reconstituted with wt platelets. Accordingly, murine (wt) PAI-1 levels in platelet lysates and releasates were 0.43 ± 0.09 ng/109 platelets and plasma concentrations equaled 0.73 ± 0.13 ng/mL. After photochemical injury, plasma PAI-1 rose to 2.9 ± 0.7 ng/mL (n = 9, P < .01). The plasma rise was prevented by ligating the carotid artery. Hence, during acute thrombosis, fibrinolysis is efficiently prevented by plasma 2-AP, but also by vascular PAI-1, locally released into the circulation after endothelial injury.

Effect of Stabilizing versus Destabilizing Interactions on Plasminogen Activator Inhibitor-1

2000 ◽  
Vol 84 (11) ◽  
pp. 871-875 ◽  
Author(s):  
Nele Vleugels ◽  
John Leys ◽  
Isabelle Knockaert ◽  
Paul Declerck
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Plasminogen Activator Inhibitor ◽  
Reactive Site ◽  
Wild Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
The Stability ◽  
Gate Region ◽  
Activator Inhibitor ◽  
Pai 1

SummaryPlasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin family, as it spontaneously converts into a latent conformation. However, the exact mechanism of this conversion is not known. Previous studies reported that neutralizing monoclonal antibodies as well as reversal or removal of charges on the s3C-s4C turn results in a destabilization of PAI-1 leading to an accelerated conversion to its latent form.In this study the effect of the reversal or removal of charges in this “gate region” (R186E/R187E, H190E/K191E, H190L/K191L and R356E) on a stable PAI-1-variant (PAI-1-stab) was investigated. Whereas PAI-1-stab has a half-life of 150 ± 66 h, PAI-1-stab-R186ER187E, PAI-1-stab-H190E-K191E, PAI-1-stab-H190L-K191L and PAI-1-stab-R356E have a strongly decreased half-life (p< 0.005 versus PAI-1-stab) of 175 ± 48 min, 75 ± 34 min, 68 ± 38 min and 79 ± 16 min, respectively. Wild-type PAI-1 (wtPAI-1) had a half-life of 55 ± 19 min. These data indicate that the stabilization induced by the mutated residues in PAI-1-stab is counteracted by the additional mutations, resulting in half-lives similar to that of wtPAI-1, thereby suggesting that the stabilizing and destabilizing forces act mainly independently in these mutants. Extrapolation of these data to other (stable) serpins leads to the hypothesis that the s3C-s4C turn and the distal hinge region of the reactive site loop plays a role for the stability of serpins in general.

scholarly journals Plasminogen Activator Inhibitor-Type I Gene Deficient Mice Show Reduced Influx of Neutrophils in Ventilator-Induced Lung Injury

2011 ◽  
Vol 2011 ◽  
pp. 1-11
Author(s):  
Esther K. Wolthuis ◽  
Alexander P. J. Vlaar ◽  
Jorrit-Jan H. Hofstra ◽  
Joris J. T. H. Roelofs ◽  
Vivian de Waard ◽  
...  
Keyword(s):  
Lung Injury ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Lavage Fluid ◽  
Type I ◽  
Wild Type ◽  
Ventilator Induced Lung Injury ◽  
Deficient Mice ◽  
Activator Inhibitor ◽  
Pai 1

Ventilator-induced lung injury (VILI) is associated with inhibition of the fibrinolytic system secondary to increased production of plasminogen activator inhibitor- (PAI-)1. To determine the role of PAI-1 on pulmonary coagulopathy and inflammation during mechanical ventilation, PAI-1 gene-deficient mice and their wild-type littermates were anesthetized (control), or anesthetized, tracheotomized and subsequently ventilated for 5 hours with either low tidal volumes () or high tidal volumes (). VILI was assessed by pulmonary coagulopathy, lung wet-to-dry ratios, total protein level in bronchoalveolar lavage fluid, neutrophil influx, histopathology, and pulmonary and plasma cytokine levels. Ventilation resulted in pulmonary coagulopathy and inflammation, with more injury following ventilation with as compared to . In PAI-1 gene-deficient mice, the influx of neutrophils in the pulmonary compartment was attenuated, while increased levels of pulmonary cytokines were found. Other endpoints of VILI were not different between PAI-1 gene-deficient and wild-type mice. These data indicate that a defect fibrinolytic response attenuates recruitment of neutrophils in VILI.

The upstream stimulatory factor-2a inhibits plasminogen activator inhibitor-1 gene expression by binding to a promoter element adjacent to the hypoxia-inducible factor-1 binding site

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2657-2666 ◽  
Author(s):  
Anatoly Samoylenko ◽  
Ulrike Roth ◽  
Kurt Jungermann ◽  
Thomas Kietzmann
Keyword(s):  
Plasminogen Activator ◽  
Hypoxia Inducible Factor ◽  
Plasminogen Activator Inhibitor ◽  
Wild Type ◽  
Upstream Stimulatory Factor ◽  
Plasminogen Activator Inhibitor 1 ◽  
Hypoxia Inducible Factor 1 ◽  
Stimulatory Factor ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Plasminogen activator inhibitor-1 (PAI-1) expression is induced by hypoxia (8% O2) via the PAI-1 promoter region −175/−159 containing a hypoxia response element (HRE-2) binding the hypoxia-inducible factor-1 (HIF-1) and an adjacent response element (HRE-1) binding a so far unknown factor. The aim of the present study was to identify this factor and to investigate its role in the regulation of PAI-1 expression. It was found by supershift assays that the upstream stimulatory factor-2a (USF-2a) bound mainly to the HRE-1 of the PAI-1 promoter and to a lesser extent to HRE-2. Overexpression of USF-2a inhibited PAI-1 messenger RNA and protein expression and activated L-type pyruvate kinase expression in primary rat hepatocytes under normoxia and hypoxia. Luciferase (Luc) gene constructs driven by 766 and 276 base pairs of the 5′-flanking region of the PAI-1 gene were transfected into primary hepatocytes together with expression vectors encoding wild-type USF-2a and a USF-2a mutant lacking DNA binding and dimerization activity (ΔHU2a). Cotransfection of the wild-type USF-2a vector reduced Luc activity by about 8-fold, whereas cotransfection of ΔHU2a did not influence Luc activity. Mutation of the HRE-1 (−175/−168) in the PAI-1 promoter Luc constructs decreased USF-dependent inhibition of Luc activity. Mutation of the HRE-2 (−165/−158) was less effective. Cotransfection of a HIF-1α vector could compete for the binding of USF at HRE-2. These results indicated that the balance between 2 transcriptional factors, HIF-1 and USF-2a, which can bind adjacent HRE sites, appears to be involved in the regulation of PAI-1 expression in many clinical conditions.

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

2006 ◽  
Vol 95 (05) ◽  
pp. 857-864 ◽  
Author(s):  
Anneke Hecke ◽  
Hilary Brooks ◽  
Matthieu Meryet-Figuière ◽  
Stephanie Minne ◽  
Stavros Konstantinides ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Small Interfering Rna ◽  
Vascular Endothelial Cells ◽  
Plasminogen Activator Inhibitor ◽  
Vascular Endothelial ◽  
Sirna Treatment ◽  
Interfering Rna ◽  
Activator Inhibitor ◽  
Pai 1

SummaryClinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissuetype 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 concentrationand 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. SiRNAmediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis.

The 4G/5G Sequence Polymorphism in the Promoter of Plasminogen Activator Inhibitor-1 (PAI-1) Gene: Relationship to Plasma PAI-1 Level in Venous Thromboembolism

1998 ◽  
Vol 79 (05) ◽  
pp. 975-979 ◽  
Author(s):  
Mojca Stegnar ◽  
Pavel Uhrin ◽  
Polona Peternel ◽  
Alenka Mavri ◽  
Barbara Salobir-Pajnič ◽  
...  
Keyword(s):  
Body Mass Index ◽  
Venous Thromboembolism ◽  
Plasminogen Activator ◽  
Body Mass ◽  
Plasminogen Activator Inhibitor ◽  
Healthy Controls ◽  
Metabolic Factors ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

SummaryImpaired fibrinolysis due to increased plasminogen activator inhibitor-1 (PAI-1) is observed in up to 40% of patients with venous thromboembolism and might be causally related to the disease. There is evidence that genetic variations in the promoter of the PAI-1 gene and metabolic factors contribute to increased plasma PAI-1 levels.A single nucleotide insertion/deletion (4G/5G) polymorphism in the promoter region of the PAI-1 gene and metabolic factors were studied in 158 unrelated patients below the age of 61 years (43 ± 11 years, mean ± standard deviation) with history of objectively confirmed venous thromboembolism and in 145 apparently healthy controls.Patients had on average two times higher PAI activity (11.9 vs. 6.1 IU/ml) and by 40% higher PAI-1 antigen (14.8 vs. 10.7 ng/ml) than healthy controls, and also higher body mass index, lipid levels, fasting glucose and insulin. Patients differed significantly from healthy controls neither in the frequency of the 4G and 5G alleles (0.57/0.43 in patients and 0.52/0.48 in controls) nor in the distribution of the 4G/5G genotypes. Possession of the 4G/4G or the 4G/5G genotype did not increase relative risk for venous thromboembolic disease and the distribution of the 4G/5G genotypes was neither associated with recurrent nor with spontaneous disease. In patients association between the 4G/5G genotypes and PAI activity (adjusted for body mass index, triglyceride and glucose level) was observed, with the highest PAI activity values in the 4G/4G genotype (14.6 IU/ml), intermediate in the 4G/5G genotype (13.3 IU/ml) and the lowest in the 5G/5G genotype (5.2 IU/ml, all values means). Association between PAI activity and triglyceride level was the strongest in the 4G/4G genotype (correlation coefficient r = 0.47, p <0.01) and the weakest in the 5G/5G genotype (r = -0.04, not significant).In conclusion, the present case-control study shows an association between the 4G/5G polymorphism in the promoter of the PAI-1 gene and plasma PAI-1 levels in patients with venous thromboembolism. Similar distribution of the 4G/5G genotypes in patients and healthy controls suggests that this genetic variation by itself is not a major risk factor for venous thromboembolism.

Food deprivation induces adipose plasminogen activator inhibitor-1 (PAI-1) expression without accumulation of plasma PAI-1 in genetically obese and diabetic db/db mice

2007 ◽  
Vol 98 (10) ◽  
pp. 864-870 ◽  
Author(s):  
Katsutaka Oishi ◽  
Naoki Ohkura ◽  
Juzo Matsuda ◽  
Norio Ishida
Keyword(s):  
Plasminogen Activator ◽  
Food Deprivation ◽  
Fibrinolytic Activity ◽  
Plasminogen Activator Inhibitor ◽  
Mrna Levels ◽  
Wild Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Adipose Tissues ◽  
Activator Inhibitor ◽  
Pai 1

SummaryRelationships between energy intake and fibrinolytic functions have been documented in detail. We evaluated food deprivation (FD) as a means of modulating fibrinolytic activity in genetically obese and diabetic db/db mice and in their lean counterparts. Twelve hours of FD induced considerable gene expression of plasminogen activator inhibitor-1 (PAI-1) in both epididymal (3.8-fold, p<0.05) and intestinal (2.4-fold, p<0.05) adipose tissues without affecting plasma PAI-1 levels in db/db mice, whereas the FD did not affect these parameters in wild-type mice. Importantly, 24 hours of FD increased the plasma PAI-1 content in wild-type (1.9-fold, p<0.01) but not in db/db mice, although adipose PAI-1 mRNA levels were significantly increased in db/db mice. The plasma PAI-1 content significantly correlated with hepatic PAI-1 mRNA levels in wild-type (r=0.84, p<0.01) and in db/db (r=0.63, p<0.01) mice. However, plasma PAI-1 did not correlate with adipose PAI-1 expression in db/db mice, although adipose tissue in general is thought to be the principal site of PAI-1 production in obesity. Hepatic PAI-1 expression was closely correlated with serum levels of free fatty acids in wild-type (r=0.72, p<0.01), but not in db/db mice. Adipose PAI-1 expression significantly correlated with serum corticosterone levels in both genotypes (wild-type, r=0.52, p<0.05; db/db, r=0.51, p<0.01), suggesting that adipose PAI-1 expression is up-regulated by fastinginduced glucocorticoids. The present findings suggested that fasting differentially affects fibrinolytic activity in obese and lean subjects and that PAI-1 expression in the liver as well as in adipose tissues comprises an important determinant of increased risk for cardiovascular disease in obesity.

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