Induction of plasminogen activator inhibitor (PAI) by lipopolysaccharide (LPS)

1987 ◽  
Author(s):  
A M DOSNE ◽  
F DUBOR ◽  
L CHEDID
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Polymyxin B ◽  
Culture Conditions ◽  
Clot Lysis ◽  
Human Endothelial Cells ◽  
Lysis Time ◽  
Binding Factor ◽  
Activator Inhibitor

It has been shown that, under culture conditions, human endothelial cells synthetize plasminogen activator inhibitor (PAI) which neutralize urokinase and tissue plasminogen activator.Treatment of human endothelial cells with LPS (50 ngto 10 μg/ml) from S. enteritidis resulted in a dose-dependent increase in PAI production.Fibrinoenzymographic analysis showed that incubation of supernatantfrom LPS-treated cells with urokinase of low and high mol. w. (33.000 and 55.000) led to disappearance of the two urokinase lytic bands and formation of high mol. w. complexes (Mr 93.000 and 107.000). The mol. w. of the urokinase binding factor was calculated to be near 50.000. Polymyxin B and colimycin could suppress this effect of LPS. Injection of LPS (30 ng-30 yg/kg in the rat led to a considerable decrease in the fibrinolytic activity of plasma euglobulins which clot lysis time was prolonged from 55 up to morethan 240 min. This hypofibrinolytic state was associated with PAI detected in euglobulins and in plasma.Large complexes (Mr 80.000-105.000) were formed between exogenous urokinase of low and high mol. w. mixed with post LPS plasma or euglobulins. Polymyxin B and Colimycin could prevent the hypofibrinolytic response to low doses of LPS. These results suggest thatPAI generation in endotoxemia could be due in part to the direct effect of LPS on endothelium.

scholarly journals Conversion of the active to latent plasminogen activator inhibitor from human endothelial cells

Blood ◽  
1987 ◽  
Vol 70 (4) ◽  
pp. 1090-1098
Author(s):  
EG Levin ◽  
L Santell
Keyword(s):  
Endothelial Cells ◽  
Conditioned Medium ◽  
Plasminogen Activator ◽  
Active Form ◽  
Plasminogen Activator Inhibitor ◽  
Human Endothelial Cells ◽  
Inhibitor Activity ◽  
Latent Form ◽  
Activator Inhibitor ◽  
Pai 1

The plasminogen activator inhibitor from human endothelial cells (PAI- 1) exists in two forms in the culture medium: an active form that binds to and inactivates plasminogen activators and a latent form that in its native state has no anti-activator activity. Inhibitor activity associated with the latent form can be generated by treatment with protein denaturants and makes up more than 98% of the total inhibitor activity in conditioned medium. Plasminogen activator inhibitor activity is also found in cell cytosol. This inhibitor activity is stable to SDS-treatment but is not enhanced by it. We investigated the relationship between this active cell-associated inhibitor and the latent PAI-1 found in the conditioned medium. Both intracellular and extracellular inhibitors were immunoprecipitated by a monoclonal antibody produced against the latent inhibitor from HT1080 fibrosarcoma cells and electrophoresis on SDS gels of various acrylamide concentrations demonstrated that both forms had the same Mr. Incubation of cytosol inhibitor at 37 degrees C resulted in a decline in inhibitor activity with a half-life of approximately 4 hours, a rate of decline similar to that of the active PAI-1 in conditioned medium, with less than 10% of the original activity present after eight hours. This decline is accelerated at higher temperatures and is not affected by the presence of a variety of protease inhibitors. Approximately 90% of the activity can be regenerated after SDS treatment suggesting that the cell associated inhibitor, during incubation at 37 degrees C, converts to a form similar to that found in conditioned medium. Despite these similarities, the apparent Stoke's radii of the active intracellular inhibitor and the latent inhibitor in conditioned medium were significantly different with values of 2.77 nm and 2.40 nm for active and latent PAI-1, respectively. Incubation of the active form at 37 degrees C resulted in the shift of the Stoke's radius to that similar to the latent PAI-1 (2.45 nm). Thus, the active and latent PAI-1, while being immunologically similar and of the same apparent Mr, can be differentiated by their behavior on gel permeation columns. This suggests that the intracellular inhibitor is a precursor to the latent form.

REGULATION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 mRNA IN HUMAN ENDOTHELIAL CELLS

1987 ◽  
Author(s):  
E A van den Berg ◽  
E Sprengers ◽  
M Jaye ◽  
W Burgess ◽  
V W M van Hinsbergh
Keyword(s):  
Endothelial Cells ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Plasminogen Activator ◽  
Cdna Clone ◽  
Plasminogen Activator Inhibitor ◽  
Mrna Levels ◽  
Human Endothelial Cells ◽  
Activator Inhibitor ◽  
Pai 1

Cultured human endothelial cells (HEC) increase their production of plasminogen activator inhibitor (PAI-1) upon stimulation with endotoxin and IL-1, agents that are known to cause an increase in PAI-1 levels in vivo. In order to study the regulation of PAI-1 synthesis at the mRNA level, we isolated a cDNA clone for the human PAI-1 gene from an endothelial expression cDNA library in λ gt 11 by screening with a PAI-1 specific antibody. Three positive cross-hybridizing clones were isolated. The longest insert (1500 bp) was partially sequenced (1000 bp). The sequence was identical to the PAI-1 sequence recently reported by others. The identity of the cDNA clone was further confirmed by comparison with part of the amino acid sequence of PAI-1. For that purpose t-PA-PAI-1 complex was purified from HEC conditioned medium by immunoadsorption to anti-t-PA IgG, and a suitable peptide was sequenced after comparison of the HPLC elution profiles of CNBr digests of t-PA and t-PA-PAI-1 complex. The amino acid sequence (M)FRQFQADFT completely matches the sequence predicted from the cDNA sequence.By hybridization of the cDNA probe to Northern blots of total cellular RNA from human umbilical vein and artery EC (HUVEC, HUAEC), two transcripts of 2.3 and 3 kb were found. Primary HUAEC, incubated for 18 hours in growth medium, produced considerable although variable levels of PAI-1 activity and contained PAI-1 mRNA levels comparable to those found in subcultured HUAEC. When subcultured HUEC were incubated for 6 h with endotoxin, IL-1 or TNF, a 2-fold increase in PAI-1 mRNA was found with each of these mediators. Stimulation of the cells in the presence of cycloheximide resulted in a further increase of the 3 kb PAI-1 transcript. The 3’ end of this transcript contains a 75 bp AT-rich sequence. Similar 3’ AT-rich sequences have been found in mRNA’s for a number of inflammatory mediators and cellular oncogenes, and in some cases it has been shown that removal of the sequence increased mRNA stability. The influence of cyclohex-imid on the larger PAI-1 transcript might be explained by inhibition of synthesis of a specific nuclease that controls the level of mRNA’s harbouring such an AT rich sequence.

scholarly journals Inhibition by Modified Oligodeoxynucleotides of the Expression of Type-1 Plasminogen Activator Inhibitor in Human Endothelial Cells

1995 ◽  
Vol 227 (1-2) ◽  
pp. 494-499 ◽  
Author(s):  
Czeslaw S. Cierniewski ◽  
Anna Babinska ◽  
Maria Swiatkowska ◽  
Malgorzata Wilczynska ◽  
Andrzej Okruszek ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Human Endothelial Cells ◽  
Activator Inhibitor

scholarly journals Porphyromonas gingivalis Gingipains-Mediated Degradation of Plasminogen Activator Inhibitor-1 Leads to Delayed Wound Healing Responses in Human Endothelial Cells

2021 ◽  
pp. 1-14
Author(s):  
Li-Ting Song ◽  
Hiroyuki Tada ◽  
Takashi Nishioka ◽  
Eiji Nemoto ◽  
Takahisa Imamura ◽  
...  
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Porphyromonas Gingivalis ◽  
Plasminogen Activator Inhibitor ◽  
Human Endothelial Cells ◽  
Plasminogen Activator Inhibitor 1 ◽  
Delayed Wound Healing ◽  
Activator Inhibitor ◽  
Pai 1

Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is constitutively produced by endothelial cells and plays a vital role in maintaining vascular homeostasis. Chronic periodontitis is an inflammatory disease characterized by bleeding of periodontal tissues that support the tooth. In this study, we aimed to determine the role of PAI-1 produced by endothelial cells in response to infections caused by the primary periodontal pathogen Porphyromonas gingivalis. We demonstrated that P. gingivalis infection resulted in significantly reduced PAI-1 levels in human endothelial cells. This reduction in PAI-1 levels could be attributed to the proteolysis of PAI-1 by P. gingivalis proteinases, especially lysine-specific gingipain-K (Kgp). We demonstrated the roles of these degradative enzymes in the endothelial cells using a Kgp-specific inhibitor and P. gingivalis gingipain-null mutants, in which the lack of the proteinases resulted in the absence of PAI-1 degradation. The degradation of PAI-1 by P. gingivalis induced a delayed wound healing response in endothelial cell layers via the low-density lipoprotein receptor-related protein. Our results collectively suggested that the proteolysis of PAI-1 in endothelial cells by gingipains of P. gingivalis might lead to the deregulation of endothelial homeostasis, thereby contributing to the permeabilization and dysfunction of the vascular endothelial barrier.

PAI-1 Plays an Important Role in the Expression of t-PA Activity in the Euglobulin Clot Lysis by Controlling the Concentration of Free t-PA

1991 ◽  
Vol 66 (04) ◽  
pp. 474-478 ◽  
Author(s):  
Tetsumei Urano ◽  
Kenichi Sumiyoshi ◽  
Michal H Pietraszek ◽  
Yumiko Takada ◽  
Akikazu Takada
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Significant Negative Correlation ◽  
Clot Lysis ◽  
Plasminogen Activator Inhibitor 1 ◽  
Positive Correlation ◽  
Lysis Time ◽  
Euglobulin Clot Lysis Time ◽  
Activator Inhibitor ◽  
Pai 1

SummaryThe antigen levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were assayed in the plasma and in the euglobulin fraction, and their contributions to the euglobulin clot lysis time (ECLT) and t-PA activity were analyzed. Total and free PAI-1 levels in both fractions showed significant positive correlation with ECLT (p <0.001), whereas t-PA antigen level did not have a high correlation coefficient with ECLT. t-PA activity showed significant negative correlation with ECLT (p <0.001) and positive correlation with free t-PA level (p <0.001), which was calculated by the ratio of the concentrations of t-PA-PAI-1 complex and the free PAI-1. Thus free t-PA seems to dissolve the euglobulin clot and its concentration seems to be controlled by the concentration of free PAI-1. These findings were confirmed by the analyses of the effects of C1-inactivator and antibody against t-PA to regular ECLT and kaolin activated ECLT, the latter of which was only inhibited by the addition of C1-inactivator whereas the former was inhibited by anti-t-PA antibody.

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