Clot Lysis Mediated by Cultured Human Microvascular Endothelial Cells

1988 ◽  
Vol 60 (03) ◽  
pp. 463-467 ◽  
Author(s):  
Wolfgang Speiser ◽  
Elisabeth Anders ◽  
Bernd R Binder ◽  
Gert Müller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Shape Change ◽  
Umbilical Vein ◽  
Cell Types ◽  
Microvascular Endothelial Cells ◽  
Clot Lysis ◽  
Microvascular Endothelial ◽  
Umbilical Vein Endothelial Cells ◽  
Fibrin Clots

SummaryThe lysis of fibrin clots on the surface of cultured human omental tissue microvascular endothelial cells (HOTMEC) and cultured human umbilical vein endothelial cells (HUVEC) was studied. Fibrin clots were made by mixing fibrinogen, plasminogen and thrombin on the surface of both cell types. Clot lysis was seen only on the surface of HOTMEC, which were found to synthesize about 100-fold more tissue plasminogen activator (tPA) antigen than HUVEC. Clot lysis of HOTMEC could be blocked by anti-tPA IgG but was not affected by the incorporation of exogenous plasminogen activator (PAI) into the clot in concentrations (75 arbitrary units) exceeding the tPA activity (21 ± 2.5 IU) of the cells. Thus, it is likely that tPA secreted by HOTMEC is protected from inhibition by PAI in the presence of fibrin and endothelial cells. The stimulation of EC to release an excess of tPA over PAI, in contrast to the secretion of an excess of PAI over tPA found in unstimulated cells in the absence of fibrin, is obviously no prerequisite for the initiation of fibrinolysis on the surface of HOTMEC. As thrombin was used for clot formation, its influence on tPA and PAI synthesis of both cell types was investigated. In contrast to HOTMEC, which were not affected by Α-thrombin, HUVEC revealed a dose-dependent increase in tPA and PAI synthesis upon incubation with the enzyme. This increase in tPA production by HUVEC was not sufficient to lyse the clots within 48 hours. Furthermore, HUVEC. behaved differently towards thrombin as these cells in contrast to HOTMEC revealed the typical shape change reaction upon incubation with the enzyme

CULTURED HUMAN OMENTAL TISSUE MICROVASCULAR ENDOTHELIAL CELLS LYSE FIBRIN CLOTS IN CONTRAST TO UMBILICAL VEIN ENDOTHELIAL CELLS

1987 ◽  
Author(s):  
W Speiser ◽  
E Anders ◽  
G Müller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Umbilical Vein ◽  
Conditioned Media ◽  
Microvascular Endothelial Cells ◽  
Clot Lysis ◽  
Human Fibrinogen ◽  
Microvascular Endothelial ◽  
Umbilical Vein Endothelial Cells ◽  
Fibrinolytic Capacity

Previously, we have shown that cultured human omental tissue microvascular endothelial cells (HOTMEC) produce about 100-fold more tPA antigen than cultured human umbilical vein endothelial cells (HUVEC). Free plasminogen activator inhibitor (PAI) activity, but not free tissue plasminogen activator (tPA) activity was detected in the conditioned media of both cell types. In the present study, the clot lysis activities of HOTMEC and HUVEC were compared with each other. For this purpose, 2 mg/ml purified human fibrinogen and 0.2 μM purified human plasminogen were added to serum free medium (SFM) on top of a confluent cell layer. After the addition of α -thrombin (1 NIH U/ml; final concentration), a clot generated within 2 min. The clots formed on the surface of HOTMEC were lysed within 6 h, whereas clots on the surface of HUVEC were not lysed within 72 h. To find out, whether tPA is involved in the activation of the fibrinolytic system, studies were performed in the presence of antibodies against tPA or urokinase (uPA). The fibrinolytic capacity of HOTMEC could be blocked by incorporation of anti-tPA IgG into the clot. Clot lysis by HOTMEC was neither affected by the incorporation of anti-uPA IgG nor of exogenous PAI derived from conditioned medium of HUVEC. Incubation of HUVEC with 1000 μl SFM containing 1 NIH U/ml α -thrombin for 1 h and consecutive cultivation in 5% fetal calf serum for 48 h resulted in a 2.5-fold increase in tPA production. The elevated tPA synthesis was accompanied by an equal increase in PAI production so that free PAI activity, measured in conditioned media, did not change after incubation with α -thrombin. In contrast to experiments with HUVEC, tPA and PAI production of HOTMEC were not influenced by α-thrombin. From these studies it is concluded that 1. the fibrinolytic capacity of HOTMEC is stronger than that of HUVEC, 2. tPA released from endothelial cells is protected from inactivation by PAI when fibrin is present, and 3. HOTMEC do not release higher concentrations of tPA and PAI after contact with (X.-thrombin as it is known for HUVEC.

HUMAN OMENTAL TISSUE MICROVASCULAR ENDOTHELIAL CELLS (HOTMEC): ISOLATION AND NEW ASPECTS OF CHARACTERIZATION

1987 ◽  
Author(s):  
E Anders ◽  
J U Alles ◽  
U Delvos ◽  
B Pötzsch ◽  
K T Preissner ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Protein C ◽  
Umbilical Vein ◽  
Calf Serum ◽  
Plasminogen Activator Inhibitor ◽  
Microvascular Endothelial Cells ◽  
Chromogenic Assay ◽  
Microvascular Endothelial ◽  
Umbilical Vein Endothelial Cells

To study the function of microvascular endothelial cells in comparison to large vessel endothelial cells, HOTMEC were enzymatically isolated from human omental tissue and plated on petri dishes precoated with an extracellular matrix prepared from isolated fibroblasts of the same tissue or precoated with fibronectin. The culture medium was supplemented with 10% fetal calf serum; growth factors were not needed. HOTMEC were subcultured in a split ratio of 1:3 and maintained in culture for up to 3 month. Cultured HOTMEC were identified and discriminated from other non-endothelial cells by different characteristics and functions. 1. The cells demonstrated the typical polygonal shape as known for endothelial cells isolated from umbilical veins. In comparison to human umbilical vein endothelial cells (HUVEC), however, HOTMEC showed prominant nuclei with several nucleoli and presented a pronounced granulation of the perinuclear cytoplasm. 2. A monoclonal antibody specific for endothelial cells was bound to cultured HOTMEC. 3. Von Willebrand Factor (vWF) antigen was demonstrated within the cells by immunofluorescence staining; measurable amounts of vWF were only found in HUVEC in contrast to HOTMEC using an ELISA. 4. The addition of purified human protein C to HOTMEC preincubated with thrombin led to the activation of the zymogen as demonstrated by a chromogenic assay system. The kinetics of protein C activation were identical for HOTMEC and HUVEC. Tissue plasminogen activator (tPA) as well as plasminogen activator inhibitor (PAI) activity were detected in the culture supernatant of HOTMEC. After incubation period of 12 h in serum-free medium, the conditioned medium of confluent HOTMEC contained 100-fold higher levels of tPA than that of HUVEC. The data demonstrate that the cells isolated from human omental tissue have morphological as well as functional characteristics typical for endothelial cells. Furthermore, the study indicates that HOTMEC and HUVEC present quantitative differences in coagulant and fibrinolytic activities.

Induction of stress proteins in human endothelial cells by heavy metal ions and heat shock

1999 ◽  
Vol 277 (5) ◽  
pp. L1026-L1033 ◽  
Author(s):  
M. Wagner ◽  
I. Hermanns ◽  
F. Bittinger ◽  
C. J. Kirkpatrick
Keyword(s):  
Heavy Metal ◽  
Endothelial Cells ◽  
Heat Shock ◽  
Metal Ions ◽  
Heavy Metal Ions ◽  
Stress Proteins ◽  
Umbilical Vein ◽  
Cell Types ◽  
Microvascular Endothelial ◽  
Umbilical Vein Endothelial Cells

In the present study, we compared the induction of heat shock proteins (HSPs) by heat and heavy metal ions in three different endothelial cell types, namely, human umbilical vein endothelial cells, human pulmonary microvascular endothelial cells, and the cell line EA.hy 926. Our results show that especially Zn2+ and Cd2+ are inducers of 70-kDa (HSP70), 60-kDa (HSP60), 32-kDa (HSP32), and 27-kDa (HSP27) HSPs. The strength of inducibility is specific for each HSP. Ni2+ and Co2+ only show an inducible effect at very high concentrations, that is, in the clearly cytotoxic range. Furthermore, we investigated the time course of HSP expression and the involvement of heat shock factor-1. Our study demonstrates that the three endothelial cell types that were under investigation show comparable stress protein expression when treated with heavy metal ions or heat shock. The expression of stress proteins may be used as an early marker for the toxic damage of cells. This damage can be an inducer of acute respiratory distress syndrome in which microvascular endothelial lesions occur early. Our study provides evidence that human umbilical vein endothelial cells or EA.hy 926 cells, which are much more easily isolated and/or cultivated than pulmonary microvascular endothelial cells, could be used as alternative cell culture systems for studies on cellular dysfunction in the lung caused by toxic substances, certainly with respect to the expression of HSPs.

Sulfated glycoconjugates enhance CD36-dependent adhesion ofPlasmodium falciparum–infected erythrocytes to human microvascular endothelial cells

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 327-333 ◽  
Author(s):  
Christopher John McCormick ◽  
Christopher I. Newbold ◽  
Anthony R. Berendt
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cells ◽  
Dextran Sulfate ◽  
Umbilical Vein ◽  
Microvascular Endothelial Cells ◽  
Cos Cells ◽  
Ofplasmodium Falciparum ◽  
Microvascular Endothelial ◽  
Umbilical Vein Endothelial Cells ◽  
Negative Cell Line

A novel adhesive pathway that enhances the adhesion ofPlasmodium falciparum-infected erythrocytes (IEs) to endothelial cells has been identified. The sulfated glycoconjugates heparin, fucoidan, dextran sulfate 5000, and dextran sulfate 500 000 caused a dramatic increase in adhesion of IEs to human dermal microvascular endothelial cells. The same sulfated glycoconjugates had little effect on IE adhesion to human umbilical vein endothelial cells, a CD36-negative cell line. The effect was abolished by a monoclonal antibody directed against CD36, suggesting that enhanced adhesion to endothelium is dependent on CD36. No effect was observed on adhesion to purified platelet CD36 cells immobilized on plastic. The same sulfated glycoconjugates enhanced adhesion of infected erythrocytes to COS cells transfected with CD36, and this was inhibited by the CD36 monoclonal antibody. These findings demonstrate a role for sulfated glycoconjugates in endothelial adherence that may be important in determining the location and magnitude of sequestration through endogenous carbohydrates. In addition, they highlight possible difficulties that may be encountered from the proposed use of sulfated glycoconjugates as antiadhesive agents in patients with severe malaria.

Sulfated glycoconjugates enhance CD36-dependent adhesion ofPlasmodium falciparum–infected erythrocytes to human microvascular endothelial cells

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 327-333 ◽  
Author(s):  
Christopher John McCormick ◽  
Christopher I. Newbold ◽  
Anthony R. Berendt
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cells ◽  
Dextran Sulfate ◽  
Umbilical Vein ◽  
Microvascular Endothelial Cells ◽  
Cos Cells ◽  
Ofplasmodium Falciparum ◽  
Microvascular Endothelial ◽  
Umbilical Vein Endothelial Cells ◽  
Negative Cell Line

Abstract A novel adhesive pathway that enhances the adhesion ofPlasmodium falciparum-infected erythrocytes (IEs) to endothelial cells has been identified. The sulfated glycoconjugates heparin, fucoidan, dextran sulfate 5000, and dextran sulfate 500 000 caused a dramatic increase in adhesion of IEs to human dermal microvascular endothelial cells. The same sulfated glycoconjugates had little effect on IE adhesion to human umbilical vein endothelial cells, a CD36-negative cell line. The effect was abolished by a monoclonal antibody directed against CD36, suggesting that enhanced adhesion to endothelium is dependent on CD36. No effect was observed on adhesion to purified platelet CD36 cells immobilized on plastic. The same sulfated glycoconjugates enhanced adhesion of infected erythrocytes to COS cells transfected with CD36, and this was inhibited by the CD36 monoclonal antibody. These findings demonstrate a role for sulfated glycoconjugates in endothelial adherence that may be important in determining the location and magnitude of sequestration through endogenous carbohydrates. In addition, they highlight possible difficulties that may be encountered from the proposed use of sulfated glycoconjugates as antiadhesive agents in patients with severe malaria.

scholarly journals Rapid Secretion of Prestored Interleukin 8 from Weibel-Palade Bodies of Microvascular Endothelial Cells

1998 ◽  
Vol 188 (9) ◽  
pp. 1751-1756 ◽  
Author(s):  
Jon Olav Utgaard ◽  
Frode L. Jahnsen ◽  
Arne Bakka ◽  
Per Brandtzaeg ◽  
Guttorm Haraldsen
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Microvascular Endothelial Cells ◽  
Double Positive ◽  
Endothelial Cell Surface ◽  
Microvascular Endothelial ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Umbilical Cords ◽  
Willebrand Factor

Interleukin (IL)-8, a C-X-C chemokine, activates integrin-mediated adhesion of neutrophils. Presentation of IL-8 on the endothelial cell surface may promote leukocyte extravasation. We found that cultured human microvascular endothelial cells from the intestine (HIMEC) and from nasal polyps (PMEC), but not human umbilical vein endothelial cells (HUVEC), contained IL-8 in intracellular granules that coexpressed von Willebrand factor (vWf  ). This observation was corroborated by the immunohistochemical observation of double-positive granules (IL-8+vWf+) in vessels of small and large intestine, nasal mucosa, and skin, whereas umbilical cords revealed no endothelial IL-8. After treatment of HIMEC or PMEC with histamine or thrombin, a dramatic increase in supernatant IL-8 concentration was observed within 3 min, whereas no increase in IL-8 was detected in supernatants of identically treated HUVEC cultures. Histamine or thrombin treatment also caused IL-8–containing granules to rapidly disappear from HIMEC. In HUVEC, IL-8–containing granules were inducible by treatment with recombinant human IL-1β for 24 h; additional histamine treatment doubled IL-8 secretion from HUVEC in the same rapid manner observed for mucosal EC. These data suggested that IL-8 prestored in microvascular endothelial cells may provide a rapid pathway for specific activation of neutrophil adhesion at sites of acute inflammation.

scholarly journals Verocytotoxin-Induced Apoptosis of Human Microvascular Endothelial Cells

2001 ◽  
Vol 12 (4) ◽  
pp. 767-778
Author(s):  
ANTONETTA H. J. M. PIJPERS ◽  
PETRA A. VAN SETTEN ◽  
LAMBERTUS P. W. J. VAN DEN HEUVEL ◽  
KAREL J. M. ASSMANN ◽  
HENDRIKUS B. P. M. DIJKMAN ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Protein Synthesis ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Microvascular Endothelial Cells ◽  
Tnf Α ◽  
Microvascular Endothelial ◽  
Induced Apoptosis ◽  
Umbilical Vein Endothelial Cells

Abstract. The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-α (TNF-α). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-α interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-α-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.

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