Tissue Plasminogen Activator Expression in Endothelial Cells Exposed to Cyclic Strain in Vitro

1992 ◽  
Vol 1 (1) ◽  
pp. 43-50 ◽  
Author(s):  
Toshiaki Iba ◽  
Bauer E. Sumpio
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Cyclic Strain ◽  
Neutral Position ◽  
Messenger Ribonucleic Acid ◽  
Tissue Plasminogen ◽  
Pai 1

The effects of cyclic strain on the production of tissue plasminogen activator (tPA) and type 1 plasminogen activator inhibitor (PAI-1) by cultured endothelial cells (EC) were examined. Human saphenous vein EC were seeded in selective areas of culture plates with flexible membrane bottoms (corresponding to specific strain regions) and grown to confluence. Membranes were deformed by vacuum (-20 kPa) at 60 cycles/min (0.5 s strain alternating with 0.5 s relaxation in the neutral position) for 5 days. EC grown in the periphery were subjected to 7-24% strain, while cells grown in the center experienced less than 7% strain. The results show a significant increase in immunoreactive tPA production on days 1, 3 and 5 compared to day 0 in EC subjected to more than 7% cyclic strain. There was no significant elevation of tPA in the medium of EC subjected to less than 7% strain. tPA activity could only be detected in the medium of EC subjected to more than 7% cyclic strain. PAI-1 levels in the medium were not significantly different in either group. In addition, immunocytochemical detection of intracellular tPA and messenger ribonucleic acid (mRNA) expression of tPA (assessed by the reverse transcriptase polymerase chain reaction utilizing tPA specific sense and antisense primers) was significantly increased in EC subjected to more than 7% cyclic strain. We conclude that a 60 cycles/min regimen of strain that is greater than 7% can selectively stimulate tPA production by EC in vitro and may contribute to the relative nonthrombogenicity of the endothelium in vivo.

Uptake and Degradation of Tissue Plasminogen Activator in Rat Liver

1988 ◽  
Vol 59 (03) ◽  
pp. 474-479 ◽  
Author(s):  
Monica Einarsson ◽  
Bård Smedsrød ◽  
Håkan Pertoft
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Rat Liver ◽  
Liver Cells ◽  
Terminal Phase ◽  
Tissue Plasminogen ◽  
Liver Endothelial Cells ◽  
Parenchymal Cells

SummaryThe mechanism of uptake of tissue plasminogen activator (tPA) in rat liver was studied. Radio-iodinated tPA was removed from the circulation after intravenous administration in a biphasic mode. The initial half life, t1/2(α), and the terminal phase, t1/2(β), were determined to be 0.5 min and 7.5 min, resp. Separation of the liver cells by collagenase perfusion and density centrifugation, revealed that the uptake per cell was two to three times higher in the non-parenchymal cells than in the parenchymal cells.Endocytosis of fluorescein isothiocyanate-labelled or 125I-labelled tPA was studied in pure cultures of liver cells in vitro. Liver endothelial cells and parenchymal cells took up and degraded tPA. Endocytosis was more efficient in liver endothelial cells than in parenchymal cells, and was almost absent in Kupffer cells.Competitivb inhibition experiments showing that excess unlabelled tPA could compete with the uptake and degradation of 125I-tPA, suggested that liver endothelial cells and parenchymal cells interact with the activator in a specific manner. Endocytosis of trace amounts of 125I-tPA in cultures of liver endothelial cells and parenchymal cells was inhibited by 50% in the presence of 19 nM unlabelled tPA. Agents that interfere with one or several steps of the endocytic machinery inhibited uptake and degradation of 125I-tPA in both cell types.These findings suggest that 1) liver endothelial cells and parenchymal cells are responsible for the rapid hepatic clearance of intravenously administered tPA; 2) the activator is taken up in these cells by specific endocytosis, and 3) endocytosed tPA is transported to the lysosomes where it is degraded.

scholarly journals Tumor necrosis factor increases the production of plasminogen activator inhibitor in human endothelial cells in vitro and in rats in vivo

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1467-1473 ◽  
Author(s):  
VW van Hinsbergh ◽  
T Kooistra ◽  
EA van den Berg ◽  
HM Princen ◽  
W Fiers ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Blood Platelets ◽  
Interleukin 1 ◽  
Plasminogen Activator Inhibitor ◽  
Activator Inhibitor ◽  
Necrosis Factor ◽  
Pai 1

Abstract The vascular endothelium plays an important role in fibrinolysis by producing tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI). The monokine tumor necrosis factor (human recombinant TNF) increased the production of PAI by cultured human endothelial cells from umbilical vein (twofold) and from foreskin microvessles (four to eight fold). This was demonstrated by titration of endothelial cell-conditioned medium with t-PA, by reverse fibrin autography, and by immunoprecipitation of [35S]PAI-1 by anti-PAI-1 IgG. TNF also induced a marked increase of PAI-1 messenger RNA (mRNA) in the cells. The stimulation of PAI activity by TNF was seen at 4 U/mL and reached a maximum at 500 U/mL. Human recombinant lymphotoxin and interleukin-1 (alpha and beta) also stimulated the production of PAI activity, while interleukin-6 was ineffective. Separate additions of TNF or interleukin-1 (IL-1) at optimal concentrations (500 U/mL and 5 U/mL, respectively) resulted in a comparable stimulation of PAI production by endothelial cells. The simultaneous addition of both mediators resulted in an additive effect. The effect of TNF could not be prevented by the addition of polymyxin B or by anti-IL-1 antibodies. Therefore, it is unlikely that TNF acts through the induction of IL-1 secretion by endothelial cells. Two hours after a bolus injection of 250,000 U/kg TNF into rats, a fivefold increase in circulating PAI levels was found. In the next ten hours, the levels returned to normal. Blood platelets do not significantly contribute to the increase in circulating PAI, because the number of platelets did not change after TNF injection and the amount of PAI in blood platelets is not sufficient for several hours during an increase in PAI activity. The acute phase reactants, fibrinogen and alpha 2-antiplasmin in rat plasma, were altered little if any two to 24 hours after injection of 250,000 U/kg TNF. In vitro, TNF did not change PAI production by human and rat hepatocytes in primary monolayer culture. Therefore, it is most likely that vascular endothelial cells contribute to the increased amount of circulating PAI induced by TNF in vivo. This increase in PAI activity might decrease fibrinolysis.

Effect of PAI-1 levels on the molar concentrations of active tissue plasminogen activator (t-PA) and t-PA/PAI-1 complex in plasma

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 930-937 ◽  
Author(s):  
WL Chandler ◽  
SL Trimble ◽  
SC Loo ◽  
D Mornin
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Molar Ratio ◽  
Molar Activity ◽  
Activity Measurements ◽  
Tissue Plasminogen ◽  
Activator Inhibitor Type ◽  
Pai 1

We determined the in vivo molar concentrations of active tissue plasminogen activator (t-PA), active plasminogen activator inhibitor type 1 (PAI-1), and t-PA/PAI-1 complex. t-PA activity was measured in plasma stabilized by immediate acidification. PAI-1 activity and t- PA/PAI-1 complex antigen were measured in citrated plasma; these measurements were corrected for the loss in PAI-1 activity and increase in complex that occurs in unacidified plasma samples due to the continued reaction between t-PA and PAI-1 after the sample was drawn. To convert t-PA and PAI-1 activity measurements into molar concentrations we determined the specific molar activity of t-PA and PAI-1 in vivo: 4.48 x 10(13) IU/mol. Of 72 subjects studied, 13 had less than 150 pmol/L active PAI-1; in these individuals 33% +/- 21% of their t-PA was active and the molar ratio of active t-PA to active PAI- 1 was 0.20 +/- 0.13. In the 11 subjects with greater than 500 pmol/L active PAI-1, 1.5% = 1.1% of the t-PA was active and the molar ratio of active t-PA to active PAI-1 was 0.0043 +/- 0.0036. Overall, the fraction of active t-PA declined exponentially as a function of the active PAI-1 concentration. During the day, the percentage of total t- PA that was active increased from 12% at 8:00 AM to 31% at 8:00 PM, while the molar ratio of active t-PA to active PAI-1 increased from 0.05 to 0.22 from morning to evening (n = 12).

scholarly journals Tissue Plasminogen Activator Coating on Implant Surfaces Reduces Staphylococcus aureus Biofilm Formation

2015 ◽  
Vol 82 (1) ◽  
pp. 394-401 ◽  
Author(s):  
Jakub Kwiecinski ◽  
Manli Na ◽  
Anders Jarneborn ◽  
Gunnar Jacobsson ◽  
Marijke Peetermans ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Medical Devices ◽  
Biofilm Formation ◽  
Bacterial Biofilm ◽  
Content Type ◽  
Tissue Plasminogen

ABSTRACTStaphylococcus aureusbiofilm infections of indwelling medical devices are a major medical challenge because of their high prevalence and antibiotic resistance. As fibrin plays an important role inS. aureusbiofilm formation, we hypothesize that coating of the implant surface with fibrinolytic agents can be used as a new method of antibiofilm prophylaxis. The effect of tissue plasminogen activator (tPA) coating onS. aureusbiofilm formation was tested within vitromicroplate biofilm assays and anin vivomouse model of biofilm infection. tPA coating efficiently inhibited biofilm formation by variousS. aureusstrains. The effect was dependent on plasminogen activation by tPA, leading to subsequent local fibrin cleavage. A tPA coating on implant surfaces prevented both early adhesion and later biomass accumulation. Furthermore, tPA coating increased the susceptibility of biofilm infections to antibiotics.In vivo, significantly fewer bacteria were detected on the surfaces of implants coated with tPA than on control implants from mice treated with cloxacillin. Fibrinolytic coatings (e.g., with tPA) reduceS. aureusbiofilm formation bothin vitroandin vivo, suggesting a novel way to prevent bacterial biofilm infections of indwelling medical devices.

scholarly journals Tumor necrosis factor increases the production of plasminogen activator inhibitor in human endothelial cells in vitro and in rats in vivo

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1467-1473
Author(s):  
VW van Hinsbergh ◽  
T Kooistra ◽  
EA van den Berg ◽  
HM Princen ◽  
W Fiers ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Blood Platelets ◽  
Interleukin 1 ◽  
Plasminogen Activator Inhibitor ◽  
Activator Inhibitor ◽  
Necrosis Factor ◽  
Pai 1

The vascular endothelium plays an important role in fibrinolysis by producing tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI). The monokine tumor necrosis factor (human recombinant TNF) increased the production of PAI by cultured human endothelial cells from umbilical vein (twofold) and from foreskin microvessles (four to eight fold). This was demonstrated by titration of endothelial cell-conditioned medium with t-PA, by reverse fibrin autography, and by immunoprecipitation of [35S]PAI-1 by anti-PAI-1 IgG. TNF also induced a marked increase of PAI-1 messenger RNA (mRNA) in the cells. The stimulation of PAI activity by TNF was seen at 4 U/mL and reached a maximum at 500 U/mL. Human recombinant lymphotoxin and interleukin-1 (alpha and beta) also stimulated the production of PAI activity, while interleukin-6 was ineffective. Separate additions of TNF or interleukin-1 (IL-1) at optimal concentrations (500 U/mL and 5 U/mL, respectively) resulted in a comparable stimulation of PAI production by endothelial cells. The simultaneous addition of both mediators resulted in an additive effect. The effect of TNF could not be prevented by the addition of polymyxin B or by anti-IL-1 antibodies. Therefore, it is unlikely that TNF acts through the induction of IL-1 secretion by endothelial cells. Two hours after a bolus injection of 250,000 U/kg TNF into rats, a fivefold increase in circulating PAI levels was found. In the next ten hours, the levels returned to normal. Blood platelets do not significantly contribute to the increase in circulating PAI, because the number of platelets did not change after TNF injection and the amount of PAI in blood platelets is not sufficient for several hours during an increase in PAI activity. The acute phase reactants, fibrinogen and alpha 2-antiplasmin in rat plasma, were altered little if any two to 24 hours after injection of 250,000 U/kg TNF. In vitro, TNF did not change PAI production by human and rat hepatocytes in primary monolayer culture. Therefore, it is most likely that vascular endothelial cells contribute to the increased amount of circulating PAI induced by TNF in vivo. This increase in PAI activity might decrease fibrinolysis.

scholarly journals Effect of PAI-1 levels on the molar concentrations of active tissue plasminogen activator (t-PA) and t-PA/PAI-1 complex in plasma

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 930-937 ◽  
Author(s):  
WL Chandler ◽  
SL Trimble ◽  
SC Loo ◽  
D Mornin
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Molar Ratio ◽  
Molar Activity ◽  
Activity Measurements ◽  
Tissue Plasminogen ◽  
Activator Inhibitor Type ◽  
Pai 1

Abstract We determined the in vivo molar concentrations of active tissue plasminogen activator (t-PA), active plasminogen activator inhibitor type 1 (PAI-1), and t-PA/PAI-1 complex. t-PA activity was measured in plasma stabilized by immediate acidification. PAI-1 activity and t- PA/PAI-1 complex antigen were measured in citrated plasma; these measurements were corrected for the loss in PAI-1 activity and increase in complex that occurs in unacidified plasma samples due to the continued reaction between t-PA and PAI-1 after the sample was drawn. To convert t-PA and PAI-1 activity measurements into molar concentrations we determined the specific molar activity of t-PA and PAI-1 in vivo: 4.48 x 10(13) IU/mol. Of 72 subjects studied, 13 had less than 150 pmol/L active PAI-1; in these individuals 33% +/- 21% of their t-PA was active and the molar ratio of active t-PA to active PAI- 1 was 0.20 +/- 0.13. In the 11 subjects with greater than 500 pmol/L active PAI-1, 1.5% = 1.1% of the t-PA was active and the molar ratio of active t-PA to active PAI-1 was 0.0043 +/- 0.0036. Overall, the fraction of active t-PA declined exponentially as a function of the active PAI-1 concentration. During the day, the percentage of total t- PA that was active increased from 12% at 8:00 AM to 31% at 8:00 PM, while the molar ratio of active t-PA to active PAI-1 increased from 0.05 to 0.22 from morning to evening (n = 12).

Plasma from patients with cirrhosis increases tissue plasminogen activator release from vascular endothelial cells in vitro

2008 ◽  
Vol 18 (3) ◽  
pp. 186-190 ◽  
Author(s):  
Takeshi Hayashi ◽  
Asahi Kamogawa ◽  
Shinsei Ro ◽  
Kai Yamaguchi ◽  
Yutaro Kobayashi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Tissue Plasminogen

scholarly journals Binding of tissue plasminogen activator to human endothelial cells. Importance of the B-chain as a ligand

1992 ◽  
Vol 287 (2) ◽  
pp. 407-413 ◽  
Author(s):  
X F Cheng ◽  
O Bäck ◽  
T K Nilsson ◽  
E Nylander Lundqvist ◽  
G Pohl ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Active Site ◽  
Binding Sites ◽  
Human Umbilical Cord ◽  
Cultured Endothelial Cells ◽  
Tissue Plasminogen ◽  
A Chain ◽  
Pai 1

The aim of the present study was to investigate the binding of tissue plasminogen activator (tPA) to cultured endothelial cells and to characterize binding structures present in the cultures. Studies on the binding of 125I-tPA to cultured endothelial cells from human umbilical-cord veins (HUVEC) indicated that the number of sites for specific binding of tPA is 8 x 10(5) per cell. Treatment with an excess of antibodies against plasminogen-activator inhibitor type 1 (PAI-1) caused an 80% decrease in the binding, leaving about 1.6 x 10(5) unoccupied binding sites per cell, which appeared to be different from PAI-1. About 1.9 x 10(5) binding sites/cell for tPA were found on the surface of HUVEC that had been detached from the matrix. This indicates that only minor amounts of PAI-1 occur on the surface of the cells. In addition, immunocytochemical analysis showed that PAI-1 antigen is present almost exclusively in the cytoplasm but was not observed on the surface of the cells, whereas tPA antigen is abundant on the plasma membrane of tPA-treated cells as well as intracellularly. Competition studies using unlabelled compounds showed that native tPA and tPA B-chain (the proteinase domain), as well as the inactive derivatives, B-chain inactivated with D-Phe-Pro-Arg-chloromethane and tPA-PAI-1 complex, caused a considerable quenching of the binding of 125I-tPA to HUVEC, whereas the isolated A-chain had no demonstrable effect. Two components (apparent molecular masses 38 kDa and 56 kDa) reacting with tPA but lacking PAI-1 antigen determinants were identified. Thus the data suggest that tPA binds to HUVEC by two principally different mechanisms. One is mediated by PAI-1, which binds and inactivates tPA with a functional active site. The other binding is achieved by components which react with sites on the activator molecule other than structures of the A-chain or the active site.

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