Rat Plasminogen Activator Inhibitor-1 Research Articles

MAPK/AP-1-dependent regulation of PAI-1 gene expression by TGF-β in rat mesangial cells

Receptor-regulated Smads and/or mitogen-activated protein kinases (MAPKs) are involved in transforming growth factor-beta (TGF-beta)-induced expression of various genes, including plasminogen activator inhibitor-1 (PAI-1). Because the sequence of the promoter region in rat PAI-1 gene differs from that in the human gene, we examined the mechanisms of TGF-beta-induced rat PAI-1 expression in rat mesangial cells. TGF-beta1-induced PAI-1 and c-fos mRNA expressions were determined by Northern blot analysis. Activation of MAPKs and Smad proteins was evaluated by an immunoblot analysis. DNA binding activities of nuclear protein were examined by using an electrophoretic mobility shift assay (EMSA). The activities of PAI-1 promoter were measured by a luciferase reporter assay. Extracellular-regulated kinase (ERK) and c-Jun NH-terminal kinase (JNK) phosphorylation, c-fos mRNA expression, and activator protein-1 (AP-1) DNA binding activity stimulated by TGF-beta1 were completely suppressed by the ERK kinase (MEK) inhibitors. EMSA and reporter analysis revealed that an AP-1-like sequence located in the proximal region of the rat PAI-1 promoter was the target for TGF-beta1, and the disruption of this AP-1-like sequence suppressed basal and TGF-beta1-induced promoter activation. TGF-beta1 also stimulated nuclear translocation of Smads and binding to palindromic Smad binding element (SBE) located in the rat PAI-1 promoter, without being affected by MEK inhibitor. Point mutation and deletion of palindromic SBE did not affect TGF-beta1-induced rat PAI-1 promoter activity. Moreover, interferon-gamma (IFN-gamma) inhibited TGF-beta1-induced PAI-1 expression through selectively suppressing the ERK-AP-1 pathway. These results suggest that the essential requirement of MAPK/AP-1 activation for TGF-beta1-induced PAI-1 expression is unique to rat mesangial cells.

Hypoxia-inducible factor-1 and hypoxia response elements mediate the induction of plasminogen activator inhibitor-1 gene expression by insulin in primary rat hepatocytes

AbstractThe expression of the plasminogen activator inhibitor-1(PAI-1) gene is enhanced by insulin both in vivo and in various cell types. Because insulin exerts a number of its biologic activities via the phosphatidylinositol 3-kinase and protein kinase B (PI3K/PKB) signaling pathway, it was the aim of the present study to investigate the role of the PI3K/PKB pathway in the expression of the PAI-1gene and to identify the insulin responsive promoter sequences. It was shown that the induction of PAI-1 mRNA and protein expression by insulin and mild hypoxia could be repressed by the PI3K inhibitor wortmannin. Overexpression of a constitutively active PKB led to induction of PAI-1 mRNA expression and of luciferase (Luc) activity from a gene construct containing 766 bp of the rat PAI-1 promoter. Mutation of the hypoxia response elements (HRE-1 and HRE-2) in rat PAI-1 promoter, which could bind hypoxia inducible factor-1 (HIF-1), abolished the induction of PAI-1 by insulin and PKB. Insulin and the constitutive active PKB also induced Luc expression in cells transfected with the pGl3EPO-HRE Luc construct, containing 3 copies of the HRE from the erythropoietin gene in front of the SV40 promoter. Furthermore, insulin and the active PKB enhanced all 3 HIF α-subunit protein levels and HIF-1 DNA-binding activity, as shown by electrophoretic mobility shift assays (EMSAs). Thus, the insulin-dependent activation of the PAI-1 gene expression can be mediated via the PI3K/PKB pathway and the transcription factor HIF-1 binding to the HREs in the PAI-1 gene promoter.

Importance of N-Terminal Residues in Plasminogen Activator Inhibitor 1 on its Antibody Induced Latency Transition

The serpin plasminogen activator inhibitor-1 (PAI-1) is a well-known risk factor for thromboembolic and cardiovascular diseases. Many efforts have been made to reveal structure-function relationship in PAI-1, including the understanding of its unique latency transition. In this study, we describe the molecular characterization of PAI-1 neutralization by MA-159M12, a monoclonal antibody against rat PAI-1. Time-dependent inactivation of PAI-1, exposure of a trypsin cleavage site typically for the latent conformation and disappearance of elastase susceptibility revealed that MA-159M12 accelerated the active to latent, conformational transition (t1/2 120 +/- 12 min and 18 +/- 3.6 min in the absence and presence of MA-159M12, p < 0.0001). Cross-reactivity analysis of the antibody with various rat/human PAI-1 chimeras revealed that the epitope resides in alpha hA of rat PAI-1. Subsequent alanine-scanning mutagenesis and binding studies demonstrated that Pro2-Leu3-Pro4-Glu5 constitute the major residues of the epitope for MA-159M12. In conclusion, these findings demonstrate that, even though unexpected based on current knowledge on PAI-1 stability and function, interference with alpha hA results in a destabilisation of its active, inhibitory conformation. Therefore, alpha hA forms a putative target for the rational development of PAI-1 neutralizing components.

Identification of a Target Site in Plasminogen Activator Inhibitor-1 That Allows Neutralization of Its Inhibitory Properties Concomitant with an Allosteric Up-regulation of Its Antiadhesive Properties

The serpin plasminogen activator inhibitor-1 (PAI-1) has a dual function: 1) it plays an important role as a direct inhibitor of the plasminogen activation system, and 2) its interaction with the adhesive glycoprotein vitronectin suggests a role in tissue remodeling and metastasis, independent from its proteinase inhibitory properties. Unique to this serpin is the close association between its conformational and functional properties. Indeed, PAI-1 can occur in an active and a latent conformation, but both functions are exclusively present in the active conformation. We report here the epitope localization and functional effects of a monoclonal antibody (MA-124K1) that inhibits rat PAI-1 activity and simultaneously increases the binding of inactive PAI-1 to vitronectin (the affinity constant of PAI-1 for vitronectin is 2 x 10(7) m(-1) in the absence of MA-124K1 and 160 x 10(7) m(-1) in the presence of MA-124K1). To the best of our knowledge, this is the first monoclonal antibody dissociating the proteinase inhibitory properties from the vitronectin binding properties in PAI-1. Mutation of Glu(212) and/or Glu(220) in rat PAI-1 to Ala results in a strongly reduced affinity or absence of binding to MA-124K1. The three-dimensional structure of PAI-1 reveals that these residues constitute a conformational epitope close to the reactive-site loop and compatible with the effect of MA-124K1 on the inhibitory properties of PAI-1. However, the vitronectin binding site is localized at the opposite site of the molecule, indicating that the effect of MA-124K1 involves an allosteric modulation of the vitronectin binding site. Cell culture experiments revealed a significant reduction of cell attachment and migration in the presence of MA-124K1, providing evidence for the functional relevance of this antibody-mediated up-regulation of the vitronectin binding properties of PAI-1. In conclusion, a novel mechanism for interference with PAI-1 functions has been identified and is of importance in the modulation of cell migration and related events (e.g. tumor metastasis).

Elucidation of the Binding Regions of PAI-1 Neutralizing Antibodies Using Chimeric Variants of Human and Rat PAI-1

Increased levels of plasminogen activator inhibitor-1 (PAI-1), the main physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma, are a known risk factor for thromboembolic and cardiovascular diseases. The elucidation of the binding site of inhibitory monoclonal antibodies may contribute to the rational design of PAI-1 modulating therapeutics. In this study, homolog-scanning mutagenesis was used to identify the binding region of a variety of human PAI-1 inhibitory antibodies, lacking cross-reactivity with rat PAI-1. Therefore. eight chimeric human/rat PAI-1 variants, containing rat PAI-1 substitutions at the N-terminal or C-terminal end with splicing sites at positions 26, 81, 187, 277 or 327, were generated and purified. Biochemical characterization revealed that all chimeras were folded properly. Subsequently, surface plasmon resonance was used to determine the affinity of various monoclonal antibodies for these chimera. Comparative analysis of the affinity and ELISA data allowed the identification of the major binding region of the inhibitory antibodies MA-8H9D4, MA-33B8F7, MA-44E4, MA-42A2F6 and MA-56A7C10. Thus, three segments in human PAI-1 containing each at least one site involved in the neutralization of PAI-1 activity could be identified, i.e. (1) the segment from residue 81 to residue 187 (comprising alpha-helices hD, hE and hF, beta-strands s4C, s3A, s2A and s1A and the loops connecting these elements). (2) the segment between residues 277 and 327 (hI, thIs5A, s5A and s6A) and (3) the region C-terminal from amino acid 327, including the reactive site loop. The current data. together with previous data, indicate that PAI-1 contains at least four different regions that could be considered as putative targets to modulate its activity.

Inactivation of Plasminogen Activator Inhibitor-1 Accelerates Thrombolysis of a Platelet-rich Thrombus in Rat Mesenteric Arterioles

To investigate the role of active plasminogen activator inhibitor 1 (PAI-1) in the evolution of a microthrombus generated in the arteriolar microcirculation, the monoclonal antibody, 33H1F7, which transforms active PAI-1 to a tissue type plasminogen activator (t-PA) substrate, was evaluated in an arteriolar thrombosis model in the rat mesentery. Arterioles (200-300 um) were stimulated electrically to create an endothelial lesion; ADP was then perfused for 2 min to induce the formation of a platelet-rich thrombus which lysed spontaneously in 140 +/- 24 s. Two successive ADP superfusions produced comparable thrombi which lysed in comparable times. Different doses of 33H1F7 were infused to rats for 30 min and the dose which inactivates rapidly and totally active rat PAI-1 (300 microg/kg/min) was selected to be tested on the thrombosis model. Infusion of 33H I F7 beginning 10 min before the ADP application significantly reduced the lysis time in comparison to the control (123 +/- 30 s versus 169 +/- 33 s, P < 0.05, paired Student's t-test) and the cumulative thrombus area during the lysis period was decreased by 56 +/- 7%. These results demonstrate that inactivation of PAI-1 is able to accelerate lysis of a platelet-rich clot in a mesenteric arteriole of the rat. Thus active PAI-1 most likely participates to the resistance to thrombolysis in the arteriolar microcirculation and its inactivation may shorten ischemic periods after microvascular obstruction such as e.g. during cerebral stroke.

In Vivo Stimulation of Vascular Plasminogen Activator Inhibitor-1 Production by very Low-Density Lipoprotein Involves Transcription Factor Binding to a VLDL-Responsive Element

High plasma levels of plasminogen activator inhibitor-1 (PAI-1) are associated with an increased risk of cardiovascular disease. There is also a close relation between high plasma levels of PAI-1 and hypertriglyceridemia. Cell culture studies have shown that very low density lipoprotein (VLDL) increases the production and secretion of PAI-1 in endothelial cells and hepatocytes, suggesting a possible mechanism for this association. To determine whether VLDL stimulates PAI-1 production in vascular cells also in vivo, Sprague-Dawley rats were injected intravenously with 6 mg/kg of VLDL (derived from human subjects with type IV hyperlipidemia). Previous studies have demonstrated that this results in an accumulation of human VLDL in the aorta and other arteries followed by increased nuclear factor-kappa B (NF-kappaB) activation. Endothelial, but not smooth muscle cells, showed a basal PAI-1 mRNA and protein expression as assessed by in situ hybridization and immunohistochemistry, respectively. Six to twenty-four hours after the VLDL injection, lipoprotein particle accumulation was seen in the aortic wall, which was accompanied by increasing PAI-1 mRNA and protein expression in endothelial and smooth muscle cells. Within the rat PAI-1 promoter we identified a sequence located at -589 to -571 with 74% homology with the recently described VLDL responsive element in the human PAI-1 promoter and located adjacent to a 4-guanosine motif presumably corresponding to the human 4G/5G polymorphism. Transient transfection studies showed that VLDL exerts its stimulatory effects on rat PAI-1 gene expression in vascular cells by interaction with promoter sequences located within bp -656 and -505. Electrophoretic mobility shift assays showed that VLDL increases the binding of as yet incompletely characterized factors to this response element. Taken together these observations support a direct influence of VLDL on vascular PAI-1 gene expression ill vivo. This stimulation is exerted on the level of PAI-1 gene transcription, and involves transcription factor binding to a VLDL responsive element adjacent to a 4G motif within the PAI-1 promoter.

Induction of the Plasminogen Activator Inhibitor-1 Gene Expression by Mild Hypoxia Via a Hypoxia Response Element Binding the Hypoxia-Inducible Factor-1 in Rat Hepatocytes

Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of both tissue-type and urokinase-type plasminogen activators. The balance between plasminogen activators and PAI-1 plays an important role in several physiological and pathophysiological processes such as atherosclerosis or thrombosis. Because these conditions are associated with hypoxia, it was the aim of the present study to investigate the influence of low O2tension on the expression of PAI-1 mRNA and protein using primary cultured rat hepatocytes as a model system. We found that PAI-1 mRNA and protein were induced by mild hypoxia (8% O2). The hypoxia-dependent PAI-1 mRNA induction was transcriptionally regulated because it was inhibited by actinomycin D (ActD). Luciferase (LUC) reporter gene constructs driven by about 800 bp of the 5′-flanking region of the rat PAI-1 gene were transiently transfected into primary rat hepatocytes; mild hypoxia caused a 3-fold induction, which was mediated by the PAI-1 promoter region -175/-158 containing 2 putative hypoxia response elements (HRE) binding the hypoxia-inducible factor (HIF-1). Mutation of the HRE-1 (-175/-168) or HRE-2 (-165/-158) also abolished the induction by mild hypoxia. Cotransfection of a HIF-1 vector and the PAI-1–LUC constructs, as well as gel shift assays, showed that the HRE-2 of the PAI-1 promoter was most critical for induction by hypoxia and HIF-1 binding. Thus, PAI-1 induction by mild hypoxia via a HIF-1 binding HRE in the rat PAI-1 promoter appears to be the mechanism causing the increase in PAI-1 in many clinical conditions associated with O2deficiency.

Natriuretic Peptides Regulate the Expression of Tissue Factor and PAI-1 in Endothelial Cells

In the present study, we demonstrate that brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) interact with angiotensin II (Ang II) in regulative blood coagulation and fibrinolysis by suppressing the expressions of both tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) induced by Ang II. The expressions of TF and PAI-1 mRNA were analyzed by northern blotting methods, and the activities of TF on the surface of rat aortic endothelial cells (RAECs) and PAI-1 in the culture media were respectively measured by chromogenic assay. Both BNP and CNP suppressed the expressions of TF and PAI-1 mRNA induced by Ang II in a time- and concentration-dependent manner via cGMP cascade, which suppressions were accompanied by respective decrease in activities of TF and PAI-1. However, neither the expression of tissue factor pathway inhibitor (TFPI) nor tissue-type plasminogen activator (TPA) mRNA was affected by the treatment of BNP and CNP.

Suppression of plasminogen activator inhibitor 1 (PAI-1) activity levels in rats by monoclonal antibodies

Summary Background : Elevated levels of plasminogen activator inhibitor-1 (PAI-1) are associated with several important thrombotic diseases. Rat models have been shown to be suitable for in vivo investigations on thrombolysis and fibrinolysis. In the present study, monoclonal antibodies (MA) against rat PAI-1 are used to reduce PAI-1 levels in endotoxin-treated rat plasma. Results : A large panel of murine monoclonal antibodies was raised against recombinant rat PAI-1. Out of 237 monoclonal antibodies, six antibodies showed a strong inhibition towards recombinant rat PAI-1 activity in vitro resulting in 70–100% inhibition of PAI-1 activity at a 16-fold molar excess of MA over PAI-1. Relative epitope analysis revealed that this panel represented at least two different epitopes. Subsequent selection of two monoclonal antibodies (MA-124K1 and MA-159M12) and evaluation of their PAI-1 neutralizing properties in vitro in rat plasma, supplemented with recombinant rat PAI-1 and in plasma from endotoxin-treated rats, revealed that MA-124K1 was capable of inhibiting native rat PAI-1 in a plasma environment. Based on these data, MA-124K1 was selected for evaluation in in vivo experiments. Rats were pre-treated with endotoxin (0.5 mg/kg, ip, 90 min prior to MA administration) to obtain high levels of native rat PAI-1 in vivo. Levels of PAI-1 antigen and activity in plasma were measured by specific ELISAs. Injection of a single dose of MA-124K1 (3 mg/kg body weight) resulted in a ∼60% inhibition of PAI-1 activity without affecting PAI-1 antigen levels. No changes were observed upon administration of MA-32K3, a control monoclonal antibody. Conclusion : MA-124K1 may serve as a useful tool in studies on the evaluation of the role of PAI-1 in cardiovascular disease using rat models.

Antithrombotic activity of a monoclonal antibody inducing the substrate form of plasminogen activator inhibitor type 1 in rat models of venous and arterial thrombosis.

1. Elevated plasminogen activator inhibitor 1 (PAI-1) is a risk factor for thrombosis, and inhibitors of the interaction between PAI-1 and tissue plasminogen activator (t-PA) have antithrombotic and prothrombolytic activity in animals. We describe the antithrombotic effects in the rat of a monoclonal antibody (MA33H1) which converts PAI-1 to a non-inhibitory substrate. 2. The activity of MA33H1 against rat PAI-1 was confirmed using two-chain t-PA and a chromogenic substrate. MA33H1 was evaluated in rat venous (thromboplastin + stasis in the abdominal vena cava) and arterial (electric current applied to a carotid artery) thrombosis models. The effects on tail-transection bleeding time were studied. 3. MA33H1 at 100 ng ml(-1) inhibited both human (44.1%) and rat PAI-1 (49.7%). This effect was concentration-dependent. Its effect on human PAI-1 was not significantly inhibited by 1 microg ml(-1) fibrin or a approximately 7 fold molar excess of vitronectin (1 nM). Inhibition of rat PAI-1 was unchanged by fibrin, but vitronectin reduced inhibition from 0.5 nM. 4. In the venous thrombosis model, MA33H1 significantly reduced thrombus weights by 38 and 58.6% at 50 and 100 microg kg(-1) min(-1) i.v. respectively. This effect was inhibited by tranexamic acid. In the arterial model, MA33H1 significantly increased the delay to occlusive thrombus formation by 58 and 142% at 50 and 100 microg kg(-1) min(-1) i.v., and did not affect bleeding time at 300 microg kg(-1) min(-1) i.v. 5. Thus, a monoclonal antibody which transforms PAI-1 to a t-PA substrate prevents thrombus formation in the rat with no effect on bleeding time at a higher dose.

Prevention of aneurysm development and rupture by local overexpression of plasminogen activator inhibitor-1.

Arterial aneurysms exhibit a loss of elastin and an increase in the plasminogen activators urokinase plasminogen activator (u-PA) and tissue plasminogen activator (t-PA). Because u-PA, t-PA, and plasmin have a limited proteolytic activity against elastin, the role of plasminogen activators in the aneurysmal disease is unclear. To investigate this question, we overexpressed plasminogen activator inhibitor-1 (PAI-1), an inhibitor of t-PA and u-PA, in a rat model of aortic aneurysm. Guinea pig-to-rat aortic xenografts were seeded with syngeneic Fischer 344 rat smooth muscle cells retrovirally transduced with the rat PAI-1 gene (LPSN group) or the vector alone (LXSN group). Some grafts were not seeded with cells (NO group). Western blots showed increased PAI-1 in grafts from the LPSN group compared with LXSN and NO groups. All grafts in the NO group (n=8) and 40% in the LXSN group ruptured between days 4 and 14. At 4 weeks in the LXSN group, the remaining unruptured grafts (n=6) were aneurysmal (diameter increase > or =100%), whereas in the LPSN group (n=6) none of the grafts had ruptured or were aneurysmal. Elastin was preserved in the LPSN group. t-PA, the major PA expressed in the model, was decreased in the LPSN group compared with the other groups, as determined by zymography. Quantitative zymography showed decreased levels of two matrix metalloproteinases (MMPs), a 28-kD caseinase, and activated MMP-9 in the LPSN group. The blockade of plasminogen activators prevents formation of aneurysms and arterial rupture by inhibiting MMP activation.

Monoclonal Antibody-based Immunoassays for the Specific Quantitation of Rat PAI-1 Antigen and Activity in Biological Samples

Two enzyme-linked immunosorbent assays (ELISAs) for the quantitation of rat plasminogen activator inhibitor-1 (PAI-1) antigen and activity, respectively, in biological fluids were developed using monoclonal antibodies raised against recombinant rat PAI-1. These assays had a lower limit of sensitivity in plasma of 0.3 and 0.15 ng/ml, respectively. The intra-assay, inter-assay and inter-dilution coefficients of variation were 9, 14 and 9%, respectively, for the antigen assay and 8, 17 and 13%, respectively for the activity assay. Assay recoveries of recombinant rat PAI-1 (5 to 20 ng/ml) added to plasma were 73 to 88% and 89 to 93% for the antigen and the activity assay, respectively. The level of PAI-1 antigen in rat plasma was 1.8 +/- 0.9 ng/ml (mean +/- SD, n = 18), with a corresponding value of 1.0 +/- 0.5 ng/ml for PAI-1 activity. In lysed platelet-rich rat plasma PAI-1 antigen was 6.0 +/- 1.0 ng/ml (n = 8) and PAI-1 activity was 2.3 +/- 0.4 ng/ml (n = 8). Endotoxin injection (0.5 mg/kg) induced a time-dependent increase of both PAI-1 antigen and PAI-1 activity levels in rat plasma. eventually resulting in a 100- to 200-fold increase (p < 0.0001 vs. baseline). A linear correlation was found between PAI-1 antigen and activity levels in normal plasma (r = 0.63, n = 18, p < 0.01) and in plasma from endotoxin-treated rats (r = 0.90, n = 35, p < 0.001). Application of these assays for the analysis of gel filtration experiments of plasma from endotoxin-treated rats demonstrated that PAI-1 antigen eluted as two peaks (with corresponding Mr of approximately 430 kDa and 61 kDa) whereas PAI-1 activity eluted as a single peak corresponding with the high molecular weight antigen form. Thus, these unique assays allowing the specific determination of rat PAI-1 antigen and rat PAI-1 activity may constitute important tools for further investigations on the pathophysiological role of PAI-1 in a variety of experimental rat models.

Expression, purification, and characterization of recombinant rat plasminogen activator inhibitor-1

Summary Plasminogen activator inhibitor-1 (PAI-1) is a physiologically important regulatory protein of the fibrinolytic system. To study in vivo its influence on a variety of biological events (thrombosis, atherosclerosis, tumour progression) a number of different experimental models in various animals, including rats, have been established. A major drawback of these models is the lack of the purified endogenous proteins and/or specific reagents for their detection in each particular species. In this study we describe the expression, in Escherichia coli (E. coli) cells, the purification, and the characterization of rat PAI-1. As expected, sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed a protein with an apparent molecular mass of ∼45 kDa. Recombinant rat PAI-1 had a specific inhibitory activity of 434 000±74 000 U/mg (mean±SD, n=11) towards human tissue-type plasminogen activator (t-PA), corresponding to 58±10% of the theoretical maximum value. Evaluation of the reaction products formed upon interaction between recombinant rat PAI-1 and its target proteinases t-PA or urokinase-type plasminogen activator revealed the presence of large amounts of covalent complex, small amounts of cleaved PAI-1 and residual latent PAI-1. Active recombinant rat PAI-1 converted spontaneously to the latent form with a half-life of 2±0.2h (n=6). The second-order rate constant of inhibition of human t-PA by recombinant rat PAI-1 was two-fold lower compared to that by recombinant human PAI-1 (0.6±0.02×107M−1s−1 vs 1.4±0.14×107M−1s−1, respectively, P The current data demonstrate the biochemical equivalence between rat PAI-1 and human PAI-1. The availability of purified recombinant rat PAI-1 will allow the development of research tools required to evaluate the in vivo role of PAI-1 in various rat models.

XR5118, a novel modulator of plasminogen activator inhibitor-1 (PAI-1), increases endogenous tPA activity in the rat

Summary XR5118, a diketopiperazine-based low molecular weight inhibitor of plasminogen activator inhibitor-1 (PAI-1) activity, was studied ex vivo and in vivo in the rat to determine whether inhibition of PAI-1 activity resulted in increased fibrinolysis and protection against thrombus formation. XR5118 reversed the inhibitory effects of human PAI-1 against tissue-type plasminogen activator (tPA), in an vitro amidolytic assay (S2251) with an IC50 value of 3.5 μM±0.19 μM (n=7). This activity was confirmed in in vitro fibrinolysis assays against both human and rat PAI-1 and, following intravenous administration to rats, XR5118 (1–5mg/kg) dose-dependently increased clot lysis in an ex vivo dilute blood clot lysis time (DBCLT) assay. At 5 mg/kg, XR5118 increased clot lysis by 41±1.6% (n=39, P

Presence of urokinase plasminogen activator and plasminogen activator inhibitor-1 messenger ribonucleic acids in rat endometrium during decidualization in vivo.

Rat endometrial stromal cells undergoing decidualization in vitro secrete urokinase-type plasminogen activator (uPA), and this secretion is regulated by prostaglandin E2. The present study was undertaken to determine whether uPA and plasminogen activator inhibitor-1 (PAI-1) mRNAs are expressed in vivo in the decidua of pregnant rats and in the deciduoma of "pseudopregnant" rats. Total RNA was prepared from nondecidualized and decidualized endometrial tissues at various stages of early pregnancy and examined by Northern blot analysis using specific cDNA probes for rat uPA and PAI-1. There was little uPA mRNA in the endometrium during the first 5 days of pregnancy (Day 1 = the presence of sperm in the vagina). A high level of uPA mRNA was detected on Day 7, and it declined thereafter. There was a gradual increase in PAI-1 mRNA in the decidua from Day 7 of pregnancy, reaching a peak level on Day 15 when the decidua was transformed into the maternal placenta. (RNA was not analyzed beyond Day 15 of pregnancy in this study.) In situ hybridization studies verified that uPA mRNA was present in the decidua adjacent to the implanting embryo on Day 7. Plasminogen activator inhibitor-1 mRNA was scattered in the decidualized endometrium, but greater amounts of PAI-1 mRNA were found in the fetal tissue on Day 10 of pregnancy. Northern blot analysis of RNA from the deciduoma produced in ovariectomized, steroid-treated rats by intrauterine injection of oil demonstrated a similar temporal pattern of expression of uPA mRNA; i.e., the level of uPA mRNA was highest on Day 7 and decreased thereafter. The level of PAI-1 mRNA in deciduoma was not detectable by Northern blot technique during the first 10 days of pseudopregnancy. These findings confirm that uPA mRNA is present in vivo in rat decidual cells, independent of the presence of a conceptus. By contrast, the level of PAI-1 mRNA in the uterus is probably influenced by the presence of the conceptus.

Prostaglandin E2 regulates production of plasminogen activator isoenzymes, urokinase receptor, and plasminogen activator inhibitor‐1 in primary cultures of rat calvarial osteoblasts

The bone resorbing agent, prostaglandin E2 (PGE2), was found to alter several components of the plasminogen activator (PA)/plasmin pathway in primary cultures of rat neonatal osteoblast-like cells. The mRNA and activities of both urokinase-type PA (uPA) and tissue-type PA (tPA) were enhanced by PGE2 treatment. The presence of mRNA for the uPA receptor (uPAR) has been demonstrated in these cells and steady-state levels shown to be greatly enhanced, the response being rapid and sustained for at least 24 hours. mRNA for plasminogen activator inhibitor 1 (PAI-1) was modulated in a biphasic manner, with inhibition of the constitutive level apparent at 4 hours of treatment and stimulation apparent at 12 hours and longer, while PAI-1 protein, measured by an ELISA assay for rat PAI-1, was diminished over this period. Neither PAI-2 mRNA nor mRNA for the broad spectrum protease inhibitor, protease nexin-1 (PN-1), was found to be modulated by PGE2. Therefore, PGE2 is likely to stimulate cell surface proteolytic activity, since uPA mRNA and cell-associated activity were elevated, as was mRNA for the cellular receptor for uPA. Although it was not possible to measure uPAR number and affinity it seems likely that elevated uPAR mRNA would translate into increased uPARs which would localize the increased uPA activity to the pericellular region. tPA mRNA and activity were also increased transiently with the activity inhibited with prolonged incubations, apparently by PAI-1. Elevation of tPA mRNA and activity may result in elevated activity within the extracellular matrix as tPA has been reported to associate with several matrix proteins. Thus the early effect of PGE2 would be to promote proteolysis, both pericellularly and in the extracellular matrix. The inhibition of PAI-1 mRNA and protein, which would contribute to the elevation of activity, is due to PGE2, but the later stimulatory effect on PAI-1 mRNA may be due to feedback regulation by transforming growth factor beta (TGF beta), secreted by osteoblasts and activated by elevated levels of PA.

Glucocorticoid regulation of the rat plasminogen activator inhibitor-1 (PAI-1) gene in FTO-2b hepatoma cells is mediated through a complex GRE

Glucocorticoid regulation of the rat plasminogen activator inhibitor-1 (PAI-1) gene in FTO-2b hepatoma cells is mediated through a complex GRE

Mechanism of glucocorticoid induction of the rat plasminogen activator inhibitor-1 gene in HTC rat hepatoma cells: identification of cis-acting regulatory elements.

Type 1 plasminogen activator inhibitor (PAI-1) is the major physiological inhibitor of plasminogen activation, inhibiting both tissue- and urokinase-type plasminogen activators. In HTC rat hepatoma cells, glucocorticoids increase PAI-1 activity, antigen and mRNA accumulation 3- to 5-fold; this increase is due solely to an increase in the rate of PAI-1 gene transcription. We have identified the cis-acting sequences in the 5'-flanking sequence of the HTC PAI-1 gene that mediate this induction. Analysis of a series of hybrid genes containing various portions of the PAI-1 5'-flanking region fused to the chloramphenicol acetyltransferase reporter gene transfected into HTC cells localized the region involved in the transcriptional regulation by glucocorticoids to between -1237 and -764. Electrophoretic mobility shift assays and DNase-I protection assays showed that a glucocorticoid response element (GRE) 15-mer located at -1212 bound the glucocorticoid receptor DNA-binding domain protein in a concentration-dependent manner. Mutations created within this GRE eliminated its ability both to confer a glucocorticoid response and to bind the glucocorticoid receptor. When placed upstream of a heterologous promoter in either orientation, this GRE conferred glucocorticoid inducibility. We, therefore, conclude that the sole cis-acting sequence required for the glucocorticoid response of the PAI-1 gene in rat HTC hepatoma cells is the GRE at -1212.

Mechanism of glucocorticoid induction of the rat plasminogen activator inhibitor-1 gene in HTC rat hepatoma cells: identification of cis- acting regulatory elements

Mechanism of glucocorticoid induction of the rat plasminogen activator inhibitor-1 gene in HTC rat hepatoma cells: identification of cis- acting regulatory elements