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.

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.

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.

Abstract 1331: Myocardial Function Alterations Correlate to Cardiac Fibrosis and Upregulation of Profibrotic Signaling in Plasminogen Activator Inhibitor-1 Deficient Mice

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
William Bradham ◽  
Linda Gleaves ◽  
Mousumi Medda ◽  
Douglas Vaughan
Keyword(s):  
Growth Factor ◽  
Plasminogen Activator ◽  
Cardiac Fibrosis ◽  
Plasminogen Activator Inhibitor ◽  
Rt Pcr ◽  
Plasminogen Activator Inhibitor 1 ◽  
Internal Dimension ◽  
Functional Consequences ◽  
Activator Inhibitor ◽  
Pai 1

Cardiac fibrosis is a common sequelae of cardiac injury and has deleterious functional consequences impacting cardiac filling, function and rhythm. Plasminogen activator inhibitor-1 (PAI-1) has been implicated in the pathogenesis of tissue fibrosis in mice. To investigate the longitudinal effect of PAI-1 on cardiac structure and function, M-mode echocardiography was employed to examine cardiac function in PAI-1 deficient (PAI-1 −/− ) and wild-type (WT) control mice in four age groups (6,12,18, 24 months). Eighteen month old PAI-1 −/− mice exhibited reduced left ventricular (LV) diastolic internal dimension ( p =0.0118) and a trend towards increased LV posterior wall (LVPW) thickness, compared to WT. Two year old PAI-1 −/− mice showed increased diastolic and systolic LVPW thickness ( p =0.0127 and p =0.0212, respectively), reduced diastolic and systolic LV internal dimension ( p =0.0486 and p =0.0124), but with preserved LV fractional shortening compared to WT. Histological examination of cardiac sections revealed fibrosis on the anterior epicardial surface of the hearts in 18 month old PKO, which in 26 month old mice had become confluent with extensive (10 –17% by area) epicardial, perivascular, and interstitial distribution (compared to none in WT). Real time polymerase chain reaction (RT-PCR) revealed upregulation of transforming growth factor beta (TGF-β) and fibroblast growth factor 2 in PAI-1 −/− compared to WT ( p =0.0234 and p =0.037, respectively). Immunofluoresence confirmed this finding with bright TGF-β staining localized in the media of intra-myocardial arterioles, and phosphorylated SMAD2/3, the downstream TGF-β signaling mediator, in areas of fibrosis. Thoracic aortic cells from aged (18 –24 month) PKO and WT mice were grown in culture, with RT-PCR revealing 4 fold increased TGF-β and 17 fold increased SMAD3 ( p <0.05 for both) RNA levels in PAI-1 −/− , supplying additional evidence for upregulation of a profibrotic TGF-β/SMAD tissue signaling pathway. The present study is one of the first to elucidate some of the functional consequences and relevant molecular signaling pathways related to aging and PAI-1 deficiency mediated cardiac fibrosis.

scholarly journals Fibroblast-specific plasminogen activator inhibitor-1 depletion ameliorates renal interstitial fibrosis after unilateral ureteral obstruction

2019 ◽  
Vol 34 (12) ◽  
pp. 2042-2050 ◽  
Author(s):  
Lan Yao ◽  
M Frances Wright ◽  
Brandon C Farmer ◽  
Laura S Peterson ◽  
Amir M Khan ◽  
...  
Keyword(s):  
Growth Factor ◽  
Plasminogen Activator ◽  
Ureteral Obstruction ◽  
Interstitial Fibrosis ◽  
Plasminogen Activator Inhibitor ◽  
Unilateral Ureteral Obstruction ◽  
Tubular Epithelium ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Background Plasminogen activator inhibitor-1 (PAI-1) expression increases extracellular matrix deposition and contributes to interstitial fibrosis in the kidney after injury. While PAI-1 is ubiquitously expressed in the kidney, we hypothesized that interstitial fibrosis is strongly dependent on fibroblast-specific PAI-1 (fbPAI-1). Methods Tenascin C Cre (TNC Cre) and fbPAI-1 knockdown (KD) mice with green fluorescent protein (GFP) expressed within the TNC construct underwent unilateral ureteral obstruction and were sacrificed 10 days later. Results GFP+ cells in fbPAI-1 KD mice showed significantly reduced PAI-1 expression. Interstitial fibrosis, measured by Sirius red staining and collagen I western blot, was significantly decreased in fbPAI-1 KD compared with TNC Cre mice. There was no significant difference in transforming growth factor β (TGF-β) expression or its activation between the two groups. However, GFP+ cells from fbPAI-1 KD mice had lower TGF β and connective tissue growth factor (CTGF) expression. The number of fibroblasts was decreased in fbPAI-1 KD compared with TNC Cre mice, correlating with decreased alpha smooth muscle actin (α-SMA) expression and less fibroblast cell proliferation. TNC Cre mice had decreased E-cadherin, a marker of differentiated tubular epithelium, in contrast to preserved expression in fbPAI-1 KD. F4/80-expressing cells, mostly CD11c+/F4/80+ cells, were increased while M1 macrophage markers were decreased in fbPAI-1 KD compared with TNC Cre mice. Conclusion These findings indicate that fbPAI-1 depletion ameliorates interstitial fibrosis by decreasing fibroblast proliferation in the renal interstitium, with resulting decreased collagen I. This is linked to decreased M1 macrophages and preserved tubular epithelium.

scholarly journals Insulin-Like Growth Factor Binding Protein-5 Binds to Plasminogen Activator Inhibitor-I*

Endocrinology ◽  
1997 ◽  
Vol 138 (7) ◽  
pp. 2972-2978 ◽  
Author(s):  
Taek Jeong Nam ◽  
Walker Busby ◽  
David R. Clemmons
Keyword(s):  
Amino Acids ◽  
Extracellular Matrix ◽  
Amino Acid ◽  
Growth Factor ◽  
Heparan Sulfate ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Insulin-like growth factor binding protein-5 (IGFBP-5) has been shown to bind to the extracellular matrix (ECM) of both fibroblasts and smooth muscle cells. The ECM-IGFBP-5 interaction is mediated in part by binding to heparan sulfate containing proteoglycans. Because proteoglycans may not be the only components of ECM that bind to IGFBP-5, we have determined its ability to bind to other ECM proteins. When a partially purified mixture of the proteins that were present in fibroblast conditioned medium was purified by IGFBP-5 affinity chromatography, a 55-kDa protein was eluted. Amino acid sequencing of the amino terminal 28 amino acids showed that it was human plasminogen activator inhibitor-1 (PAI-1). To determine if this interaction was specific, purified human PAI-1 was incubated with IGFBP-5 and the IGFBP-5/PAI-1 complex immunoprecipitated with anti-PAI-1 antiserum. When the precipitate was analyzed by immunoblotting using anti-IGFBP-5 antiserum, the intensity of the IGFBP-5 band was substantially increased compared with controls that did not contain human PAI-1. A synthetic IGFBP-5 peptide that contained the amino acid sequence between positions 201 and 218 inhibited IGFBP-5/PAI-1 interaction. Coincubation of IGFBP-5 mutants that contained substitutions for specific basic residues located between positions 201 and 218 with PAI-1 indicated that some of these amino acids were important for binding. Two mutants that contained neutral substitutions for specific basic amino acids within the glycosaminoglycan binding domain had reduced binding to PAI-1. In contrast, three other mutants that also had substitutions for charged residues in the same region had no reduction in binding. Heparin and heparan sulfate inhibited the IGFBP-5/PAI-1 interaction; however, several other glycosaminoglycans had no effect. PAI-1 was determined to be an important ECM component for binding because approximately 27% of total ECM binding could be inhibited with anti-PAI-1 antiserum. Competitive binding studies with unlabeled IGFBP-5 showed that the dissociation constant of PAI-1 for IGFBP-5 was 9.1 × 10−8m. In summary, IGFBP-5 binds specifically to plasminogen activator inhibitor-1. Because this is present in the extracellular matrix of several cell types, it may be one of the important binding components of ECM. PAI-1 binding partially protects IGFBP-5 from proteolysis, suggesting that it is one of the ECM components that is involved in mediating this effect.

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