The Effects of Insulin-Like Growth Factor-1 on Plasminogen Activator Inhibitor-1 Synthesis and Secretion: Results from In Vitro and In Vivo Studies

1993 ◽  
Vol 70 (06) ◽  
pp. 1009-1013 ◽  
Author(s):  
S J Padayatty ◽  
S Orme ◽  
P D Zenobi ◽  
M H Stickland ◽  
P E Belchetz ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Mean Value ◽  
Diabetic Patients ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

SummaryIn vitro studies have shown that insulin and IGF-1 releases the fibrinolytic inhibitor plasminogen activator inhibitor-1 (PAI-1) from cells of hepatic origin. To investigate the effects of IGF-1 on fibrinolysis: 1) cultured hepatoma cells were grown in the presence of IGF-1 and media collected for secreted PAI-1 and cells probed for PAI-1 mRNA, 2) 8 hypopituitary patients were treated with recombinant human growth hormone (rhGH) and 3) 5 type 2 diabetic patients were treated with recombinant human IGF-1 (rhIGF-1). Treatment of Hep G2 cells with IGF-1 (1000 ng/ml) increased secretion of PAI-1 from a median value of 80 ng/106 cells (range 21-91) to 144 ng/106 cells (range 128-169) after 24 h (p <0.01). Synthesis of PAI-1 mRNA increased in a similar fashion. Treatment of hypopituitary patients with rhGH led to an increase in circulating IGF-1 from a mean value of 166 (range 41-324) ng/ml at baseline to 322 (77-575) ng/ml at 4 weeks and 259 (104-533) ng/ml after 8 weeks (p <0.02). Despite this, no changes in circulating PAI-1 or fibrinolysis occurred. Type II diabetic patients treated with rhIGF-1 showed an increase in circulating IGF-1 from a mean value of 120 ng/ml (range 109-196), at baseline to 823 ng/ml (585-894) after 5 days. This also was not associated with changes in circulating PAI-1 or in fibrinolysis. The results confirm that IGF-1 induces the synthesis of PAI-1 in Hep G2 cells. However, marked increases in IGF-1 had no effect on circulating PAI-1 or fibrinolysis.

scholarly journals Endocytosis and lysosomal delivery of tissue plasminogen activator- inhibitor 1 complexes in Hep G2 cells

Blood ◽  
1992 ◽  
Vol 80 (11) ◽  
pp. 2746-2754 ◽  
Author(s):  
DM Underhill ◽  
DA Owensby ◽  
PA Morton ◽  
AL Schwartz
Keyword(s):  
Plasminogen Activator ◽  
Culture Media ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Plasminogen Activator Inhibitor 1 ◽  
G2 Cells ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Receptor-mediated endocytosis of tissue-type plasminogen activator (t- PA)-plasminogen activator inhibitor type 1 (PAI-1) complexes results in their clearance by Hep G2 cells. After complexes are internalized, the t-PA component is degraded. However, neither the locus of intracellular catabolism nor the fate of PAI-1 has been elucidated. To characterize these aspects of t-PA-PAI-1 catabolism, the subcellular distribution of a prebound cohort of ligand molecules was delineated after internalization at 37 degrees C. 125I-t-PA.PAI-1 and t-PA.125I-PAI-1 were compared in separate experiments. After ligand uptake, intracellular vesicles were separated on density gradients. Internalized 125I-t-PA.PAI-1 concentrated initially in endosomes. After 20 minutes of uptake, the complex began to appear in lysosomes. Subsequently, low molecular weight labeled ligand fragments were detected in culture media. A panel of lysosomotropic agents, including primaquine, chloroquine, ammonium chloride, and a combination of leupeptin and pepstatin A, inhibited degradation. When t-PA.125I-PAI-1 rather than 125I-t-PA.PAI-1 was internalized, strikingly different results were observed. Although the kinetics of internalization and the intracellular itinerary were indistinguishable for the differently labeled complexes, the 125I-PAI-1 component of t-PA.125I-PAI-1 resisted rapid degradation. After a rapid loss of t-PA, the 125I-PAI-1 moiety persisted in lysosomes for up to 180 minutes. Thus, internalized t- PA.PAI-1 is targeted to lysosomes in which PAI-1 is relatively more stable than t-PA.

scholarly journals Endocytosis and lysosomal delivery of tissue plasminogen activator- inhibitor 1 complexes in Hep G2 cells

Blood ◽  
1992 ◽  
Vol 80 (11) ◽  
pp. 2746-2754
Author(s):  
DM Underhill ◽  
DA Owensby ◽  
PA Morton ◽  
AL Schwartz
Keyword(s):  
Plasminogen Activator ◽  
Culture Media ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Plasminogen Activator Inhibitor 1 ◽  
G2 Cells ◽  
Activator Inhibitor ◽  
Pai 1

Receptor-mediated endocytosis of tissue-type plasminogen activator (t- PA)-plasminogen activator inhibitor type 1 (PAI-1) complexes results in their clearance by Hep G2 cells. After complexes are internalized, the t-PA component is degraded. However, neither the locus of intracellular catabolism nor the fate of PAI-1 has been elucidated. To characterize these aspects of t-PA-PAI-1 catabolism, the subcellular distribution of a prebound cohort of ligand molecules was delineated after internalization at 37 degrees C. 125I-t-PA.PAI-1 and t-PA.125I-PAI-1 were compared in separate experiments. After ligand uptake, intracellular vesicles were separated on density gradients. Internalized 125I-t-PA.PAI-1 concentrated initially in endosomes. After 20 minutes of uptake, the complex began to appear in lysosomes. Subsequently, low molecular weight labeled ligand fragments were detected in culture media. A panel of lysosomotropic agents, including primaquine, chloroquine, ammonium chloride, and a combination of leupeptin and pepstatin A, inhibited degradation. When t-PA.125I-PAI-1 rather than 125I-t-PA.PAI-1 was internalized, strikingly different results were observed. Although the kinetics of internalization and the intracellular itinerary were indistinguishable for the differently labeled complexes, the 125I-PAI-1 component of t-PA.125I-PAI-1 resisted rapid degradation. After a rapid loss of t-PA, the 125I-PAI-1 moiety persisted in lysosomes for up to 180 minutes. Thus, internalized t- PA.PAI-1 is targeted to lysosomes in which PAI-1 is relatively more stable than t-PA.

Up-Regulated Expression of Plasminogen Activator Inhibitor-1 in Hep G2 Cells: Interrelationship between Insulin and Insulin-Like Growth Factor 1

1995 ◽  
Vol 73 (02) ◽  
pp. 268-274 ◽  
Author(s):  
F Anfosso ◽  
M C Alessi ◽  
G Nalbone ◽  
N Chomiki ◽  
M Henry ◽  
...  
Keyword(s):  
Growth Factor ◽  
Plasminogen Activator ◽  
Insulin Receptors ◽  
Plasminogen Activator Inhibitor ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Plasminogen Activator Inhibitor 1 ◽  
G2 Cells ◽  
Activator Inhibitor ◽  
Pai 1

SummaryInsulin resistance represents a situation with a high risk of athero-thrombosis and is accompanied by increased plasma plasminogen activator inhibitor-1 (PAI-1) levels. Fasting insulin level is highly correlated with PAI-1 levels in plasma. It has been shown that insulin increases PAI-1 synthesis by the human hepatoma cell line Hep G2. Moreover when Hep G2 cells expressing a down-regulation of insulin receptors by incubation with 10-7 M insulin, were stimulated by 10-9 M insulin, an overexpression of PAI-1 synthesis was observed despite a reduced number of insulin receptors. Insulin-like growth factor 1 (IGF-1) shares many properties with insulin. The aim of the present study was to evaluate the effect of IGF-1 on PAI-1 synthesis by Hep G2 cells down-regulated either by insulin or IGF-1.Incubation of Hep G2 cells with increasing doses from 10-9 to 10-7 M IGF-1 induced a dose-dependent stimulation of PAI-1 synthesis up to 4.5-fold the control level. When cells were first pre-incubated with 10-7M IGF-1 for 18 h, acid washed, and then stimulated with 10-9 M IGF-1, the expression of IGF-1 receptors was greatly reduced (up to 70%). In contrast PAI-1 secretion was increased 3.4-fold the level of control cells and by 1.9-fold the level of cells first stimulated with 10-9M IGF-1. Both transcripts of PAI-1 mRNA were also increased. The overexpression of PAI-1 synthesis was observed irrespective of the hormone used in the down-regulation step (i.e. 10-9 M insulin or IGF-1) or in the stimulation step (i. e. 10-9 M insulin or IGF-1). The results showed an interrelationship between insulin and IGF-1 on PAI-1 synthesis in down-regulated Hep G2 cells. They also suggest that in the insulin resistant state, IGF-1 would be able to participate in the increase in PAI-1 plasma levels by stimulating down-regulated insulin target cells.

The Roles of α2-Antiplasmin and Plasminogen Activator Inhibitor 1 (PAI-1) in the Inhibition of Clot Lysis

1993 ◽  
Vol 70 (02) ◽  
pp. 301-306 ◽  
Author(s):  
Linda A Robbie ◽  
Nuala A Booth ◽  
Alison M Croll ◽  
Bruce Bennett
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Dependent Inhibition ◽  
Activator Inhibitor ◽  
Pai 1

SummaryThe relative importance of the two major inhibitors of fibrinolysis, α2-antiplasmin (α2-AP) and plasminogen activator inhibitor (PAI-1), were investigated using a simple microtitre plate system to study fibrin clot lysis in vitro. Cross-linked fibrin clots contained plasminogen and tissue plasminogen activator (t-PA) at concentrations close to physiological. Purified α2-AP and PAI-1 caused dose-dependent inhibition. All the inhibition due to normal plasma, either platelet-rich or poor, was neutralised only by antibodies to α2-AP. Isolated platelets, at a final concentration similar to that in blood, 2.5 × 108/ml, markedly inhibited clot lysis. This inhibition was neutralised only by antibodies to PAI-1. At the normal circulating ratio of plasma to platelets, α2-AP was the dominant inhibitor. When the platelet:plasma ratio was raised some 20-fold, platelet PAI-1 provided a significant contribution. High local concentrations of PAI-1 do occur in thrombi in vivo, indicating a role for PAI-1, complementary to that of α2-AP, in such situations.

Localization of a Vitronectin Binding Region of Plasminogen Activator Inhibitor-1

1995 ◽  
Vol 73 (05) ◽  
pp. 829-834 ◽  
Author(s):  
Jaya Padmanabhan ◽  
David C Sane
Keyword(s):  
Binding Site ◽  
Plasminogen Activator ◽  
Inhibitory Activity ◽  
Plasminogen Activator Inhibitor ◽  
Structural Homology ◽  
Binding Region ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

SummaryThe PAI-1 binding site for VN was studied using two independent methods. PAI-1 was cleaved by Staph V8 protease, producing 8 fragments, only 2 of which bound to [125I]-VN. These fragments were predicted to overlap between residues 91-130. Since PAI-2 has structural homology to PAI-1, but does not bind to vitronectin, chimeras of PAI-1 and PAI-2 were constructed. Four chimeras, containing PAI-1 residues 1-70,1-105,1-114, and 1-167 were constructed and expressed in vitro. PAI-1, PAI-2, and all of the chimeras retained inhibitory activity for t-PA, but only the chimera containing PAI-1 residues 1-167 formed a complex with VN. Together, these results predict that the VN binding site of PAI-1 is between residues 115-130.

Abstract T P75: Temporal Profiles of Plasminogen Activator Inhibitor-1 Concentration and Activity Changes in Circulation after Focal Stroke and Tissue Type Plasminogen Activator Administration in Rats

Stroke ◽  
2014 ◽  
Vol 45 (suppl_1) ◽  
Author(s):  
Qi Liu ◽  
Xiang Fan ◽  
Helen Brogren ◽  
Ming-Ming Ning ◽  
Eng H Lo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Temporal Profiles ◽  
Activator Inhibitor ◽  
Pai 1

Aims: Plasminogen activator inhibitor-1 (PAI-1) is the main and potent endogenous tissue-type plasminogen activator (tPA) inhibitor, but an important question on whether PAI-1 in blood stream responds and interferes with the exogenously administered tPA remains unexplored. We for the first time investigated temporal profiles of PAI-1 concentration and activity in circulation after stroke and tPA administration in rats. Methods: Permanent MCAO focal stroke of rats were treated with saline or 10mg/kg tPA at 3 hours after stroke (n=10 per group). Plasma (platelet free) PAI-1 antigen and activity levels were measured by ELISA at before stroke, 3, 4.5 (1.5 hours after saline or tPA treatments) and 24 hours after stroke. Since vascular endothelial cells and platelets are two major cellular sources for PAI-1 in circulation, we measured releases of PAI-1 from cultured endothelial cells and isolated platelets after direct tPA (4 μg/ml) exposures for 60 min in vitro by ELISA (n=4 per group). Results: At 3 hours after stroke, both plasma PAI-1 antigen and activity were significantly increased (3.09±0.67, and 3.42±0.57 fold of before stroke baseline, respectively, all data are expressed as mean±SE). At 4.5 hours after stroke, intravenous tPA administration significantly further elevated PAI-1 antigen levels (5.26±1.24), while as expected that tPA neutralized most elevated PAI-1 activity (0.33±0.05). At 24 hours after stroke, PAI-1 antigen levels returned to the before baseline level, however, there was a significantly higher PAI-1 activity (2.51±0.53) in tPA treated rats. In vitro tPA exposures significantly increased PAI-1 releases into culture medium in cultured endothelial cells (1.65±0.08) and platelets (2.02±0.17). Conclution: Our experimental results suggest that tPA administration may further elevate stroke-increased blood PAI-1 concentration, but also increase PAI-1 activity at late 24 hours after stroke. The increased PAI-1 releases after tPA exposures in vitro suggest tPA may directly stimulate PAI-1 secretions from vascular walls and circulation platelets, which partially contributes to the PAI-1 elevation observed in focal stroke rats. The underlying regulation mechanisms and pathological consequence need further investigation.

Fibrate-modulated Expression of Fibrinogen, Plasminogen Activator Inhibitor-1 and Apolipoprotein A-I in Cultured Cynomolgus Monkey Hepatocytes

1998 ◽  
Vol 80 (12) ◽  
pp. 942-948 ◽  
Author(s):  
M. Kockx ◽  
H. M. G. Princen ◽  
T. Kooistra
Keyword(s):  
Plasminogen Activator ◽  
Cynomolgus Monkey ◽  
Plasma Concentrations ◽  
Plasminogen Activator Inhibitor ◽  
Peroxisome Proliferator ◽  
Plasminogen Activator Inhibitor 1 ◽  
Apolipoprotein A ◽  
Activator Inhibitor ◽  
Pai 1

SummaryFibrates are used to lower plasma triglycerides and cholesterol levels in hyperlipidemic patients. In addition, fibrates have been found to alter the plasma concentrations of fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and apolipoprotein A-I (apo A-I). We have investigated the in vitro effects of fibrates on fibrinogen, PAI-1 and apo A-I synthesis and the underlying regulatory mechanisms in primary monkey hepatocytes.We show that fibrates time- and dose-dependently increase fibrinogen and apo A-I expression and decrease PAI-1 expression in cultured cynomolgus monkey hepatocytes, the effects demonstrating different potency for different fibrates. After three consecutive periods of 24 h the most effective fibrate, ciprofibrate (at 1 mmol/l), increased fibrinogen and apo A-I synthesis to 356% and 322% of control levels, respectively. Maximum inhibition of PAI-1 synthesis was about 50% of control levels and was reached by 1 mmol/l gemfibrozil or ciprofibrate after 48 h. A ligand for the retinoid-X-receptor (RXR), 9-cis retinoic acid, and specific activators of the peroxisome proliferator-activated receptor-α (PPARα), Wy14,643 and ETYA, influenced fibrinogen, PAI-1 and apo A-I expression in a similar fashion, suggesting a role for the PPARα/RXRα heterodimer in the regulation of these genes. When comparing the effects of the various compounds on PPARα trans-activation activity as determined in a PPARα-sensitive reporter gene system and the ability of the compounds to affect fibrinogen, PAI-1 and apo A-I antigen production, a good correlation (r = 0.80; p <0.01) between PPARα transactivation and fibrinogen expression was found. Apo A-I expression correlated only weakly with PPARα transactivation activity (r = 0.47; p = 0.24), whereas such a correlation was absent for PAI-1 (r = 0.03; p = 0.95). These results strongly suggest an involvement of PPARα in the regulation of fibrinogen gene expression.

scholarly journals Plasminogen activator inhibitor-1 secretion of endothelial cells increases fibrinolytic resistance of an in vitro fibrin clot: evidence for a key role of endothelial cells in thrombolytic resistance

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4204-4213 ◽  
Author(s):  
S Handt ◽  
WG Jerome ◽  
L Tietze ◽  
RR Hantgan
Keyword(s):  
Endothelial Cells ◽  
Blood Vessels ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Time Dependent ◽  
Plasminogen Activator Inhibitor 1 ◽  
Necrosis Factor Alpha ◽  
Activator Inhibitor ◽  
Pai 1

Time-dependent thrombolytic resistance is a critical problem in thrombolytic therapy for acute myocardial infarction. Platelets have been regarded as the main source of plasminogen activator inhibitor-1 (PAI-1) found in occlusive platelet-rich clots. However, endothelial cells are also known to influence the fibrinolytic capacity of blood vessels, but their ability to actively mediate time-dependent thrombolytic resistance has not been fully established. We will show that, in vitro, tumor necrosis factor-alpha-stimulated endothelial cells secrete large amounts of PAI-1 over a period of hours, which then binds to fibrin and protects the clot from tissue plasminogen activator- induced fibrinolysis. In vivo, endothelial cells covering atherosclerotic plaques are influenced by cytokines synthesized by plaque cells. Therefore, we propose that continuous activation of endothelial cells in atherosclerotic blood vessels, followed by elevated PAI-1 secretion and storage of active PAI-1 in the fibrin matrix, leads to clot stabilization. This scenario makes endothelial cells a major factor in time-dependent thrombolytic resistance.

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