scholarly journals Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators.

1982 ◽  
Vol 94 (3) ◽  
pp. 631-636 ◽  
Author(s):  
E G Levin ◽  
D J Loskutoff
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Degradation Products ◽  
Tissue Type ◽  
Freezing And Thawing ◽  
Prolonged Incubation ◽  
Aortic Endothelial Cells ◽  
Cell Extracts ◽  
Bovine Endothelial Cells ◽  
Repeated Freezing

Cell extracts and conditioned media (CM) from cultured bovine aortic endothelial cells (BAEs) were fractionated by PAGE in the presence SDS, and plasminogen activator (PA) activity was localized by fibrin autography. Multiple molecular weight forms of PA were detected in both preparations. Cell-associated PAs had Mr of 48,000, 74,000, and 100,000 while secreted PAs showed Mr of 52,000, 74,000, and 100,000. A broad zone of activity (Mr 80,000-100,000) also was present in both cellular fractions. In addition, PAs of Mr 41,000 and 30,000 appeared upon prolonged incubation or repeated freezing and thawing of the samples, and probably represent degradation products of higher molecular weight forms. This complex lysis pattern was not observed when CM was subjected to isoelectric focusing. Instead, only two classes of activator were resolved, one at pH 8.5, the other at 7.6. Analysis of focused samples by SDS PAGE revealed that the activity at pH 8.5 resulted exclusively from the Mr 52,000 form; all other forms were recovered at pH 7.6. The activity of the Mr 52,000 form was neutralized by anti-urokinase IgG but was not affected by antitissue activator IgG indicating that it is a urokinaselike PA. The activities of the Mr 74,000-100,000 forms were not affected by anti-urokinase. They were blocked by antitissue activator suggesting that all the forms in this group were tissue-type PAs. The multiple forms of PA were differentially sensitive to inactivation by diisopropylfluorophosphate (DFP). Treatment of CM with 10 mM DFP for 2 h at 37 degrees C only partially inhibited the 52,000-dalton form. However, it completely inactivated the 74,000-dalton partially inhibited the 52,000-dalton form. However, it completely inactivated the 74,000-dalton PA. The activity of the Mr 100,000 form was not affected by this treatment, or by treatment with 40 mM DFP. Thus, cultured BAEs produce multiple, immunologically distinct forms of PA which differ in size, charge, and sensitivity to DFP. These forms include both urokinaselike and tissue-activator-like PAs. The possibility that one of these forms is a zymogen is discussed.

Low Molecular Weight Fibrinogen Degradation Products Stimulate the Release of Growth Factors from Endothelial Cells

1992 ◽  
Vol 68 (03) ◽  
pp. 357-363 ◽  
Author(s):  
Roberto Lorenzet ◽  
Joan H Sobel ◽  
Alessandra Bini ◽  
Larry D Witte
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Growth Factors ◽  
Degradation Products ◽  
Cell Damage ◽  
Low Molecular Weight ◽  
Aortic Endothelial Cells ◽  
Fibrinogen Degradation Products ◽  
3T3 Fibroblasts ◽  
Growth Assay

SummaryCultured porcine aortic endothelial cells (PAEC) constitutively produce and secrete in their culture medium mitogens collectively called endothelial cell-derived growth factors (EDGFs). Incubation of PAEC with fibrinogen-degradation products (FDPs) obtained by plasmin digestion of highly purified fibrinogen caused an increased release of EDGFs, as assessed by [3H]-thymidine incorporation in 3T3 mouse fibroblasts. The effect was time-dependent and correlated with the degree of fibrinogenolysis. It was accompanied by elongation of the cells. Neither increase in EDGFs release nor cell damage was observed when non-degraded fibrinogen was incubated with endothelial cells. Low molecular weight fibrinogen degradation products (LMWFDPs) (M r ≤ 10,000), and the higher molecular weight fibrinogen fragments D and E were tested under the same conditions. Only the LMWFDPs caused elongation and damage to PAEC and a marked stimulation (up to 12 fold) of EDGFs release. A low density growth assay revealed that the released EDGFs were mitogenically active on the same PAEC. The activity of the released EDGFs was time and dose dependent on both 3T3 fibroblasts and PAEC, indicating that LMWFDPs caused enhanced release of EDGFs that can act in paracrine and autocrine fashion. This study suggests an additional role for fibrinogenolysis contributing to wound healing, and possibly to atherosclerosis.

Multiple Molecular Forms Of Plasminogen Activator Produced By Cultured Bovine Endothelial Cells

1981 ◽  
Author(s):  
Eugene G Levin ◽  
David J Loskutoff
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Molecular Forms ◽  
Cell Extracts ◽  
Fresh Serum ◽  
Vessel Patency ◽  
Bovine Endothelial Cells ◽  
Overlay Technique

The production of plasminogen activator (PA) by the vascular endothelium has been implicated in the maintenance of vessel patency. Cultured bovine aortic endothelial (BAE) cells were employed to study and compare the cell associated and secreted forms of PA. Samples were fractionated by polyacrylamide gel electrophoresis in the presence of SDS. PA activity in the gel was localized by the fibrin-overlay technique. Cellular PAs were found to be membrane associated and to consist of a major form of Mr 48,000 (C48) and minor forms of 53,000 (C53), 74,000 (C74), and 100,000 (C100). Incubation of the cell extracts at 37°C resulted in the appearance of two additional forms of Mr 41,000 and 33,000 suggesting that these forms were degradation products. Serum-free conditioned medium (CM) contained secreted PAs of Mr 52,000 (S52), 55,000 (S55), 74,000 (S74) and 100,000 (S100). In addition, a broad zone of fibrinolytic activity was observed in the region between Mr 80,000 and 95,000. Cellular PAs have isoelectric points of pH 8.5-8.6 and 7.5 while secreted PAs demonstrate activity at pH 8.6, 8.5, 8.0, and 7.5. The forms showed differential sensitivities to DFP with S74 and C74 being inactivated by 1mM DFP within one Hr at 37°C while S52 and C48 were still partially active after treatment with 10mM for two Hrs. S100 was completely retractile to treatment with 40 mM DFP. The addition of fresh serum to confluent cultures resulted in the disappearance of C48 and C74, and of S52 and S74, but caused an increase in C100 and S100. These studies indicate that several forms of PA are produced by endothelial cells and suggest that the production of each may be independently regulated.

scholarly journals STUDIES ON OXIDATION AND REDUCTION BY PNEUMOCOCCUS

1924 ◽  
Vol 39 (3) ◽  
pp. 357-366 ◽  
Author(s):  
Oswald T. Avery ◽  
James M. Neill
Keyword(s):  
Yeast Extract ◽  
Room Temperature ◽  
Prolonged Exposure ◽  
Freezing And Thawing ◽  
Peroxide Formation ◽  
Cell Extracts ◽  
Repeated Freezing ◽  
Phosphate Solutions ◽  
Animal Tests

In the present paper methods have been described for the preparation of sterile extracts of pneumococci. These extracts may be obtained by dissolving the bacteria in broth cultures by means of bile, or by extraction of the cellular substances by repeated freezing and thawing of broth or saline suspensions of unwashed cells. Under special precautions these extracts may be passed through Berkefeld filters without loss of potency. In this procedure, as in all other manipulations incident to their preparation, the extracts should be protected as far as possible from contact with air. All extracts were proved sterile by cultural and animal tests. Sterile extracts of unwashed pneumococcus cells promptly form peroxide on exposure to air. Peroxide formation is almost as active in extracts aerated at 2°C. as in those exposed to the air at room temperature. Detectable amounts of peroxide may be produced by these cell extracts within the reaction range of pH 5 to 9, the optimal zone lying at reactions less acid than pH 6. The peroxide-forming activity of the extracts is gradually diminished by prolonged exposure to 55°C., and is completely destroyed by heating at 65°C. for 5 minutes. Cell extracts of pneumococci which have been thoroughly washed prior to extraction in salt or phosphate solutions exhibit no peroxide-forming activity. These extracts of washed cells may be activated by the addition of the cell washings, yeast extract, or muscle infusion.

Progress of fibrinolysis during tumor necrosis factor infusions in humans. Concomitant increase in tissue-type plasminogen activator, plasminogen activator inhibitor type-1, and fibrin(ogen) degradation products

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2284-2289 ◽  
Author(s):  
VW van Hinsbergh ◽  
KA Bauer ◽  
T Kooistra ◽  
C Kluft ◽  
G Dooijewaard ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tumor Necrosis Factor ◽  
Plasminogen Activator ◽  
Tumor Necrosis ◽  
Degradation Products ◽  
Tissue Type ◽  
Necrosis Factor ◽  
Pai 1

Abstract Several investigators have reported that tumor necrosis factor (TNF) can alter the production of plasminogen activator type-1 (PAI-1) and plasminogen activators (PAs) by endothelial cells in vitro. We have examined the in vivo effects of recombinant human TNF administration on fibrinolysis as assessed by parameters in plasma during a 24-hour period of continuous TNF infusion to 17 cancer patients with active disease. The plasma levels of PAI activity increased sevenfold after 3 and 24 hours of TNF infusion. This was the result of an increase of PAI- 1 antigen; PAI-2 antigen was not detectable. Plasma concentrations of tissue-type PA (t-PA) antigen increased twofold to fivefold after 3 and 24 hours of TNF infusion, whereas urokinase-type PA antigen levels in plasma remained unaltered. After 3 hours of TNF infusion the plasma levels of alpha 2-antiplasmin were slightly decreased, 5% on average, suggesting that fibrinolysis continued. After 24 hours of TNF infusion a highly significant increase in fibrin- plus fibrinogen-degradation products, and separately of fibrin degradation products and fibrinogen degradation products, was found. This indicates that fibrinolysis persisted, at least partly, in the presence of high levels of PAI activity. Whereas PAI-1 production increased, t-PA production by human endothelial cells in vitro remains unaltered or even decreases on TNF addition. It has been shown previously that TNF infusion in our patients results in thrombin and fibrin generation. Therefore, it is possible that thrombin, not TNF, is the actual stimulus for t-PA production in our patients. We speculate that fibrin is formed during TNF infusions and that plasmin is generated by t-PA action immediately on the initial formation of (soluble) fibrin molecules. Such a process may explain the generation of degradation products of both fibrin and fibrinogen during infusion of TNF in patients.

BINDING AND METABOLISM OF HEPARIN BY ENDOTHELIAL CELLS

1987 ◽  
Author(s):  
S Vannucchi ◽  
F Pasquali ◽  
P Bianchi-ni ◽  
M Ruggiero
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
De Novo ◽  
Degradation Products ◽  
Low Molecular Weight ◽  
Competition Binding ◽  
Capillary Endothelial Cells ◽  
A Site ◽  
Binding Sequence

In this study we show that bovineadrenal capillary endothelial cells(BACE) contain heparin (HP); this HP has been found associated with the cell surface (i.e; trypsin-removable^and intracellularly. How-ever, experiments with [ sjsodium sulfate labelling, demonstrate that BACE cells donot synthesize HP de novo, but they uptake it from serum. We have studied binding, uptake, and metabolism odifferent molecular weight-HPs: 13 Kd-HP from bovine source, 14 Kd-HP from porcine source, 4.5 Kd, and 2.5-HP fragments. Comparison among different HPs, was carried out by calculating the IC from competition curves for [3HJ- HP. Binding of labelled-HP to BACE cells was specificand saturable. Dextran sulfate and glycosaminoglycans did not compete for binding; only heparan sulfate showed some competition. Binding of different HPs was strictly dependent on their molecular weight; 2.5 Kd- HP was unable to bind to cells, although sulfation degree of this fragment and of unfractionated HP was almost identical. Therefore, we assume that a specific oligosaccharide sequence could be responsible for HP binding to BACE cells; this hypothetical "binding sequence" could then be lost in very low molecular weight-HP fragments. BACE cells are also able to internalize HP, and they release its low molecular weight degradation products into culture medium. Thus we suggest that endothelial cells might represent a site for the metabolism of endogenous and exogenous HP in vivo.

Redox active hemoglobin enhances lipopolysaccharide-induced injury to cultured bovine endothelial cells

2004 ◽  
Vol 287 (4) ◽  
pp. H1875-H1882 ◽  
Author(s):  
Felice D'Agnillo
Keyword(s):  
Endothelial Cells ◽  
Glucose Oxidase ◽  
Combined Treatment ◽  
Direct Interaction ◽  
Redox Activity ◽  
Bovine Hemoglobin ◽  
Free Hemoglobin ◽  
Aortic Endothelial Cells ◽  
Bovine Endothelial Cells ◽  
Induced Apoptosis

The interaction of cell-free hemoglobin with lipopolysaccharide (LPS) is thought to aggravate the pathophysiology of sepsis and/or septic shock. This study examines the possible modulatory role of cell-free hemoglobin on LPS-induced apoptosis of cultured bovine aortic endothelial cells. Experiments were performed with or without fetal bovine serum, a source of LPS-binding protein and soluble CD14. In the absence of serum, LPS alone or coincubated with purified bovine hemoglobin (BvHb), human hemoglobin (Hb), or α-cross-linked Hb (ααHb) did not induce apoptosis. In the presence of serum, LPS induced significant apoptosis. LPS combined with BvHb, Hb, or ααHb produced the same extent of apoptosis as LPS alone. To examine whether the H2O2-driven redox activity of hemoglobin alters LPS-induced apoptosis, glucose oxidase was added to the system to generate a subtoxic flux of H2O2. The combined treatment of LPS, glucose oxidase, and BvHb, Hb, or ααHb enhanced apoptosis compared with LPS alone. These findings support a possible mechanism whereby the redox cycling of hemoglobin, and not its direct interaction with LPS, contributes to the hemoglobin-mediated enhancement of LPS-related pathophysiology.

Angiotensin II Increases Plasminogen Activator Inhibitor-1 and Tissue Factor mRNA Expression without Changing that of Tissue Type Plasminogen Activator or Tissue Factor Pathway Inhibitor in Cultured Rat Aortic Endothelial Cells

1997 ◽  
Vol 77 (06) ◽  
pp. 1189-1195 ◽  
Author(s):  
Hiromi Nishimura ◽  
Hajime Tsuji ◽  
Haruchika Masuda ◽  
Katsumi Nakagawa ◽  
Yoshihumi Nakahara ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Ang Ii ◽  
Plasminogen Activator Inhibitor 1 ◽  
Aortic Endothelial Cells ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryAngiotensin converting enzyme inhibitors (ACE-I) have been reported to prevent the recurrence of cardiovascular events. The mechanism of this decrease, however, can not be completely explained by anti-hypertensive and anti-hypertrophic effects of ACE-I. To investigate the mechanism of this decrease, we studied the regulation of plasminogen activator inhibitor-1 (PAI-1), tissue type plasminogen activator (TPA), tissue factor (TF), and tissue factor pathway inhibitor (TFPI) by angiotensin II (Ang II) in cultured rat aortic endothelial cells. Ang II increased PAI-1 and TF mRNA expression without affecting that of TPA or TFPI. These inductions were accompanied by increases in PAI-1 and TF activities and were inhibited by a type 1 Ang II receptor antagonist. The results suggest that Ang II decreases the antithrombotic properties of endothelial cells which increases the chance of thrombosis. Thus, inhibition of the renin-angiotensin system may be beneficial to prevent thrombus formation in treatment of ischemic heart disease.

Binding of Fibrinogen to Cultured Bovine Endothelial Cells

1985 ◽  
Vol 53 (01) ◽  
pp. 026-031 ◽  
Author(s):  
Ulrich Delvos ◽  
Klaus T Preissner ◽  
Gert Müller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Receptor Site ◽  
Cell Interaction ◽  
Binding Properties ◽  
Aortic Endothelial Cells ◽  
Calcium Dependent ◽  
Bovine Aortic Endothelial Cells ◽  
Fibrinogen Binding ◽  
Bovine Endothelial Cells ◽  
Displacement Experiments

SummaryThe binding properties of purified bovine fibrinogen to bovine aortic endothelial cells have been examined in a tissue culture system. Endothelial cells bound 125I-fibrinogen in a calcium dependent fashion. Removal of calcium by EDTA instantaneously detached most of the cell-associated fibrinogen. Binding of fibrinogen to the endothelial cells was not saturable with time and dosage. Competition studies and displacement experiments did not indicate the involvement of a specific receptor site for the fibrinogen-endothelial cell-interaction. Bovine serum albumin provided in the physiological ratio of 20:1 to fibrinogen competed as effectively as unlabelled fibrinogen for 125I-fibrinogen binding to the endothelial cells. And furthermore, internalization of cell- associated tracer into the endothelial cells that could be demonstrated in the presence of serum free medium did not occur in the presence of albumin. These data suggest that fibrinogen binding to intact bovine endothelial cells is unspecific and presumably negligible in the presence of physiological albumin concentrations.

Stimulation Of Prostacyclin Production In Aortic Endothelial Cells By A Non-Dialysable Serum Factor

1981 ◽  
Author(s):  
E R Hall ◽  
M Rafelson ◽  
K Wu
Keyword(s):  
Endothelial Cells ◽  
Arachidonic Acid ◽  
Calf Serum ◽  
Dependent Manner ◽  
Freezing And Thawing ◽  
Concentration Dependent Manner ◽  
Aortic Endothelial Cells ◽  
Prostacyclin Production ◽  
Aortic Endothelial

The production of prostacyclin (PGI2) by vascular endothelial cells is thought to be of primary importance in maintaining normal hemostasis. We have investigated the production of prostacyclin in bovine arterial endothelial cells maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 30% fetal calf serum. Intact, confluent monolayers of endothelial cells (3x106 cells) in passages 2 through 6 were used. The growth medium was removed and the cells were washed in DMEM that did not contain serum. 3 mls of medium alone or containing normal plasma or serum was then added and incubated at 37°C for 15 min. Then, 1 mg of arachidonic acid was added and the cells incubated for an additional hour. The test medium was removed, centrifuged to remove any loose cells and stored at -70°C. To determine the production of PGI2 by the endothelial cells, the medium was assayed for 6-keto-PGF1α, the stable metabolite of PGI2, by radioimmunoassay. The synthesis of prostacyclin by bovine aortic endothelial cells was significantly increased in a concentration dependent manner by both normal platelet poor plasma and normal serum. This increase in prostacyclin production was inhibited by both aspirin and indomethacin, indicating an increase in synthesis rather than the release of PGI2. Furthermore, this increase could be demonstrated in the presence or absence of added arachidonic acid. The active component in plasma and serum was non-dialysable, eliminating the possibility of a small compound such as bradykinin or angiotensin II. This active factor was present after freezing and thawing the plasma and serum and was heat stable (60°C, 5 min). The presence of an endogenous prostacyclin stimulating factor may be significant in the in vivo regulation of prostacyclin production.

Sign in / Sign up

Export Citation Format

Share Document