The Production of Heparin Cofactor li Is Not Regulated by Inflammatory Cytokines in Human Hepatoma Cells: Comparison with Plasminogen Activator Inhibitor Type-1

1996 ◽  
Vol 75 (02) ◽  
pp. 298-302 ◽  
Author(s):  
Chisato Kolke ◽  
Yumiko Hayakawa ◽  
Kenji Niiya ◽  
Nobuo Sakuragawa ◽  
Hideo Sasaki
Keyword(s):  
Plasminogen Activator Inhibitor ◽  
Mrna Levels ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Tnf Α ◽  
G2 Cells ◽  
Inhibitor Type ◽  
Activator Inhibitor Type ◽  
Activator Inhibitor

SummaryUsing the Northern blot technique, we screened 6 human hepatoma cell lines to investigate the regulation mechanism of heparin cofactor II (HCII) biosynthesis. We found that HuH-7 and Hep G2 cells constitutively expressed the HC II gene. In conditioned medium, HuH-7 cells constantly produced HC II that was functionally active and formed a complex with thrombin in the presence of dermatan sulfate. HC II is thought be an acute phase reactant, and, therefore, we examined the effects of the major inflammatory cytokines, IL-6, IL-1β, and TNF-α, on the regulation of HC II production in HuH-7 and Hep G2 cells. In HuH-7 cells, the antigen and mRNA levels of plasminogen activator inhibitor type-1 (PAI-1), an acute phase protein produced by hepa-tocytes, were increased in response to stimulation with either IL-6 or IL-1 (3 or both, but HC II antigen and mRNA levels were not changed by the same stimulation. Even when Hep G2 cells were treated with a combination of three cytokines, IL-6, IL-1β, and TNF-α, HC II antigen and mRNA levels were not changed; however, PAI-1 antigen and mRNA levels were clearly increased. These results suggest that the production of HC II in hepatoma cells is not regulated by the major inflammatory mediators, IL-6, IL-iβ, and TNF-α.

Carbon Monoxide-Releasing Molecule-Derived CO Regulates Tissue Factor, Plasminogen Activator Inhibitor Type 1, and Thrombomodulin Production by Human Endothelial Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1146-1146
Author(s):  
Keiko Maruyama ◽  
Eriko Morishita ◽  
Shigeki Ohtake ◽  
Akihiro Yachie ◽  
Shinji Nakao
Keyword(s):  
Plasminogen Activator Inhibitor ◽  
Mapk Signaling ◽  
Mrna Levels ◽  
Protein Levels ◽  
Tnf Α ◽  
Inhibitor Type ◽  
Activator Inhibitor Type ◽  
Activator Inhibitor ◽  
Pai 1

Abstract Abstract 1146 Background and purpose: Heme oxygenase-1 (HO-1) is a cytoprotective and anti-inflammatory protein that catalyzes the conversion of heme into biliverdin, free iron, and carbon monoxide (CO). HO-1 is rapidly induced by various oxidative stresses and inflammation, thereby playing an important role in the self defence system. Recently, Yachie, et al. reported the first human case of HO-1 deficiency. This patient showed prominent signs of intravascular hemolysis, endothelial cell injury, and abnormalities in the coagulation / fibrinolysis system, suggesting the involvement of HO-1 or HO-1 products, such as CO, regulation of coagulation / fibrinolysis system. The current study examined whether tricarbonyldichlororuthenium (II) dimer (CORM-2), which liberated CO in the presence of dimethyl sulfoxide (DMSO), modulates the expression of tissue factor (TF), plasminogen activator inhibitor type 1 (PAI-1) and thrombomodulin (TM) in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were pretreated with CORM-2 at the concentration of 50 μM for 3h, washed and stimulated with tumor necrosis factor-α(TNF-α, 10 ng/ml) for additional 2 or 5h. The mRNA and protein levels of TF, PAI-1 and TM in the cultured HUVECs were determined by real-time reverse transcriptase polymerase chain reaction and Western blotting. To determine whether CORM-2 affects the MAPK signaling pathways, the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase1/2 (ERK1/2) in the HUVECs were analyzed with Western blotting. Results: After TNF-α stimulation, TF mRNA levels were approximately 6-fold and PAI-1 mRNA levels were approximately 8-fold increased, and TM mRNA levels were decreased by 20% compared to the control. Similarly to the mRNA expression, TF and PAI-1 protein levels were increased while the TM protein level was decreased. On the other hand, pretreatment of HUVECs with CORM-2 significantly decreased TF mRNA levels (approximately 80% suppression), and PAI-1 mRNA levels (approximately 90% suppression) while increased TM mRNA levels by 3-fold as compared to the TNF-α-stimulated group (p<0.05; Figure 1). Similarly, the pretreatment with CORM-2 inhibited TNF-α-induced TF and PAI-1 protein up-regulation and TM protein down-regulation. CORM-2 inhibited TNF-α-induced activation of p38MAPK and ERK1/2 by 50% compared to the control. Conclusions: These results indicate that CO liberated by CORM-2 suppresses the TNF-α-induced TF and PAI-1 up-regulation and prevents the TNF-α-induced TM down-regulation in HUVECs via MAPK signaling pathways. Thus, the modulation of endothelial function by CO may offer a novel antithrombotic option for treatment of the hypercoagulable state associated with inflammation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Synergistic induction of plasminogen activator inhibitor type-1 in HEP G2 cells by thrombin and transforming growth factor-beta

Blood ◽  
1992 ◽  
Vol 79 (1) ◽  
pp. 75-81 ◽  
Author(s):  
WE Hopkins ◽  
S Fujii ◽  
BE Sobel
Keyword(s):  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Plasminogen Activator Inhibitor ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
G2 Cells ◽  
Inhibitor Type ◽  
Activator Inhibitor Type ◽  
Pai 1

Abstract Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic modulator of the fibrinolytic system. Its activity in plasma increases in diverse thrombotic states. The large synthetic capacity of the liver make it a source of potentially large amounts of PAI-1. Because thrombin activity increases in association with thrombotic disorders and because specific binding sites for thrombin have been identified on hepatocytes, we characterized the effect of thrombin on hepatocyte PAI- 1 production. Incubation of Hep G2 cells with human alpha-thrombin resulted in a dose- and time-dependent increase in the concentration of PAI-1 in conditioned media. This effect was inhibited completely by hirudin and by antithrombin III. Steady-state levels of both the 3.2-kb and 2.2-kb forms of PAI-1 mRNA increased after stimulation of the cells with thrombin, indicating that thrombin influences PAI-1 expression in Hep G2 cells at the pretranslational level. Incubation of Hep G2 cells with alpha-thrombin and either platelet lysates or purified transforming growth factor-beta (TGF-beta), both previously shown to augment hepatocyte PAI-1 expression, resulted in a synergistic increase in the concentration of PAI-1 in conditioned media. PAI-1 mRNA appeared to be synergistically increased as well. Thus, thrombin increases expression of both PAI-1 protein and mRNA in Hep G2 cells and exerts synergistic effects with TGF-beta. These results underscore the potential importance of inhibition of thrombin under conditions in which thrombolysis is induced pharmacologically.

scholarly journals Synergistic induction of plasminogen activator inhibitor type-1 in HEP G2 cells by thrombin and transforming growth factor-beta

Blood ◽  
1992 ◽  
Vol 79 (1) ◽  
pp. 75-81
Author(s):  
WE Hopkins ◽  
S Fujii ◽  
BE Sobel
Keyword(s):  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Plasminogen Activator Inhibitor ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
G2 Cells ◽  
Inhibitor Type ◽  
Activator Inhibitor Type ◽  
Pai 1

Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic modulator of the fibrinolytic system. Its activity in plasma increases in diverse thrombotic states. The large synthetic capacity of the liver make it a source of potentially large amounts of PAI-1. Because thrombin activity increases in association with thrombotic disorders and because specific binding sites for thrombin have been identified on hepatocytes, we characterized the effect of thrombin on hepatocyte PAI- 1 production. Incubation of Hep G2 cells with human alpha-thrombin resulted in a dose- and time-dependent increase in the concentration of PAI-1 in conditioned media. This effect was inhibited completely by hirudin and by antithrombin III. Steady-state levels of both the 3.2-kb and 2.2-kb forms of PAI-1 mRNA increased after stimulation of the cells with thrombin, indicating that thrombin influences PAI-1 expression in Hep G2 cells at the pretranslational level. Incubation of Hep G2 cells with alpha-thrombin and either platelet lysates or purified transforming growth factor-beta (TGF-beta), both previously shown to augment hepatocyte PAI-1 expression, resulted in a synergistic increase in the concentration of PAI-1 in conditioned media. PAI-1 mRNA appeared to be synergistically increased as well. Thus, thrombin increases expression of both PAI-1 protein and mRNA in Hep G2 cells and exerts synergistic effects with TGF-beta. These results underscore the potential importance of inhibition of thrombin under conditions in which thrombolysis is induced pharmacologically.

Physiological testosterone stimulates tissue plasminogen activator and tissue factor pathway inhibitor and inhibits plasminogen activator inhibitor type 1 release in endothelial cellsThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual peer-review process.

2007 ◽  
Vol 85 (2) ◽  
pp. 246-251 ◽  
Author(s):  
Hong Jin ◽  
Jijin Lin ◽  
Lu Fu ◽  
Yi-Fang Mei ◽  
Geng Peng ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Mrna Levels ◽  
Tissue Plasminogen ◽  
Tissue Factor Pathway ◽  
Inhibitor Type ◽  
Activator Inhibitor Type ◽  
Activator Inhibitor ◽  
Pai 1

There is a striking gender difference in atherosclerotic vascular disease. For decades, testosterone was considered detrimental to the cardiovascular system. Recent studies, however, have presented some alternative results. The aim of this study was to evaluate the effect of testosterone, using physiological and supraphysiological concentrations, on antigen and mRNA levels of tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), and tissue factor pathway inhibitor (TFPI) released by human umbilical vein endothelial cells and to investigate the cellular mechanism. Cells within 2–3 passages were cultured in 25 cm2 flasks or plated onto 96-well plates with a density of about 1 × 105 cells/mL as recommended. The cells were incubated in the presence or absence of testosterone (3, 30, 3 × 103, 3 × 104 nmol/L) for 48 h. Levels of tPA, PAI-1, and TFPI antigen were assayed with ELISA kits. Reverse transcriptase PCR was carried out to detect tPA, PAI-1, and TFPI mRNA levels. Cells were incubated in androgen-receptor antagonist (flutamide 10 µmol/L) or aromatase inhibitor (aminoglutethimide 50 µmol/L) for 3 h, and then the experiments were repeated. Testosterone at a physiologic concentration (30 nmol/L) increased the antigen levels of tPA and TFPI significantly (P < 0.05). However, tPA and TFPI levels were markedly reduced (P < 0.05) at a larger dose (3 × 104 nmol/L). On the other hand, PAI-1 antigen levels decreased significantly at the testosterone concentrations ranging from 3 to 3 × 104 nmol/L (P < 0.05). The change in the levels of tPA and TFPI were reflected in the corresponding change in mRNA levels. Flutamide attenuated the effect of testosterone at physiological concentration (30 nmol/L). The results demonstrated that testosterone at physiological concentrations may have a beneficial influence on the haemostatic system through enhancement of anticoagulant activity, resulting from stimulation of TFPI and tPA expression and inhibition of PAI-1 secretion by the endothelium.

Mediators of Induction of Augmented Expression of Plasminogen Activator Inhibitor Type-1 in Hep G2 Cells by Platelets

1991 ◽  
Vol 66 (02) ◽  
pp. 239-245 ◽  
Author(s):  
William E Hopkins ◽  
Donald R Westerhausen ◽  
Satoshi Fujii ◽  
Joseph J Billadello ◽  
Burton E Sobel
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Plasminogen Activator Inhibitor ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
G2 Cells ◽  
Inhibitor Type ◽  
Activator Inhibitor Type ◽  
Pai 1

SummaryPlasminogen activator inhibitor type-1 (PAI-1) is a physiologic modulator of the fibrinolytic system. We have shown previously that PAI-1 biosynthesis in cultured cells depends on several factors in serum. Because platelets are richly endowed with specific growth factors and because the release reaction is an integral part of thrombosis, the present study was performed to determine whether platelets augment PAI-1 production and if so, to define mediators responsible. Hep G2 cells were used to determine whether platelet lysates increased PAI-1 synthesis in a dose and time-dependent manner. In cells labeled metabolically with 35S-methionine for 6 h, an increase in labeled PAI-1 was elicited indicative of de novo synthesis as well as increased secretion of PAI-1 mediated by platelet lysates. Steady state levels of both the 3.2 and 2.2 kb forms of PAI-1 mRNA increased after 2 h and peaked in 3-5 h in a dose-dependent fashion as well. Incubation of Hep G2 cells with collagen activated platelets resulted in a similar induction of PAI-1 mRNA. The increase in PAI-1 mRNA occurred with exposure of the cells to platelet lysates for intervals as brief as 15 min and was not inhibited by cycloheximide indicating its independence of new protein synthesis. In order to identify the factors in platelets responsible for the induction of PAI-1 synthesis in the Hep G2 cell model system, neutralizing antibodies were used to inhibit specific platelet associated growth factors. Antibodies to transforming growth factor-β (TGF-β) and to the epidermal growth factor (EGF)/transforming growth factor alpha (TGF-α) receptor inhibited the platelet lysate-mediated increase in PAI-1 protein by 77%. The results indicate that physiologic concentrations of platelet derived TGF-β and EGF/TGF-α increase PAI-1 expression in Hep G2 cells and suggest that the augmentation of PAI-1 synthesis induced by platelets is mediated by specific platelet associated growth factors that may contribute to observed increases in circulating PAI-1 levels in patients with disorders manifested by thrombosis.

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