scholarly journals Evidence that agonists stimulate bivalent-cation influx into human endothelial cells

1988 ◽  
Vol 255 (1) ◽  
pp. 179-184 ◽  
Author(s):  
T J Hallam ◽  
R Jacob ◽  
J E Merritt
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Umbilical Vein ◽  
Free Calcium ◽  
Human Endothelial Cells ◽  
Bivalent Cation ◽  
Human Umbilical Vein ◽  
Fura 2 ◽  
Bivalent Cations ◽  
Umbilical Vein Endothelial Cells

Human umbilical-vein endothelial cells stimulated with thrombin or histamine show an increase in [Ca2+]i (cytoplasmic free calcium concn.) that is maintained well above the basal pre-stimulated value as long as agonist and a source of extracellular Ca2+ are present. These results provide circumstantial evidence that agonists stimulate influx of Ca2+ across the plasma membrane and into the cytoplasm. Here, we have used Mn2+ as the extracellular bivalent cation which can bind to the fluorescent Ca2+ indicator fura-2 to quench its fluorescence completely. Human umbilical-vein endothelial cells were loaded with fura-2 and, in the presence of extracellular Mn2+, thrombin and histamine were shown to cause quenching of the intracellular dye. This result demonstrates conclusively that agonists can stimulate the influx of bivalent cations. Stimulated discharge of Ca2+ from intracellular stores and influx of Mn2+ were temporally resolved in the same cells to show that release of Ca2+ from intracellular stores clearly precedes influx. Influx of Mn2+ was also demonstrated when extracellular Mn2+ was added after agonist at a time when [Ca2+]i had fallen back to the basal value, showing that influx is not dependent on elevated [Ca2+]i.

scholarly journals Ca2+ influx does more than provide releasable Ca2+ to maintain repetitive spiking in human umbilical vein endothelial cells

1996 ◽  
Vol 320 (2) ◽  
pp. 505-517 ◽  
Author(s):  
Anthony J. MORGAN ◽  
Ron JACOB
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Inositol Trisphosphate ◽  
Free Medium ◽  
Bivalent Cation ◽  
Human Umbilical Vein ◽  
Fura 2 ◽  
Umbilical Vein Endothelial Cells ◽  
Small Subpopulation

We investigated why oscillations of intracellular Ca2+ concentrations ([Ca2+]i) in endothelial cells challenged by submaximal histamine run down in Ca2+-free medium despite stores retaining most of their Ca2+. One explanation is that only a small subpopulation of the Ca2+ stores oscillate and are completely emptied of Ca2+. To investigate if influx refills an empty store subpopulation, we differentiated between cations entering the cell and those released from internal stores by using extracellular Sr2+ as a Ca2+ surrogate; we distinguished between [Sr2+]i and [Ca2+]i by using the larger effect of Sr2+ on fura 2 fluorescence at 360 nm (F360). Ca2+ was still available for release when oscillations had run down since oscillations promptly reappeared on addition of Sr2+o and these were predominantly of Ca2+ (indicated by F360 changes). Also, totally depleting Ca2+ stores inhibited Sr2+-induced oscillations, suggesting that Sr2+ entry leads to Ca2+ release. In contrast, Ba2+o was unable to stimulate oscillations. Finally, oscillations generated by photolytic release of inositol trisphosphate (IP3) analogues were similarly sensitive to extracellular Ca2+ and Sr2+. We conclude that stores (or a subpopulation) are not completely depleted of Ca2+ when oscillations run down in Ca2+-free medium. Bivalent cation entry therefore maintains sensitivity to IP3, possibly by maintaining luminal bivalent cation levels.

A mathematical model of cytosolic calcium dynamics in human umbilical vein endothelial cells

1996 ◽  
Vol 270 (5) ◽  
pp. C1556-C1569 ◽  
Author(s):  
T. F. Wiesner ◽  
B. C. Berk ◽  
R. M. Nerem
Keyword(s):  
Mathematical Model ◽  
Endothelial Cells ◽  
Calcium Release ◽  
Umbilical Vein ◽  
Kinetic Equations ◽  
Cytosolic Calcium ◽  
Receptor Activation ◽  
Free Calcium ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

Important among the responses of endothelial cells are cytosolic free calcium transients. These transients are mediated by several factors, including blood-borne agonists, extracellular calcium, and fluid-imposed shear forces. The transients are characterized by a rapid rise followed by a plateau phase. A base mathematical model is presented that reasonably reproduces the measured calcium transient in cultured human umbilical vein endothelial cells responding to thrombin. Kinetic equations for receptor activation and calcium mobilization comprise the model. A graded response of intracellular free calcium to increasing concentrations of agonist is predicted. Also predicted is the elevation of the peak value and the plateau level by steady nonspecific leak of calcium across the plasma membrane. The influences of capacitative calcium entry, calcium-induced calcium release, and buffering by cytosolic proteins are investigated parametrically. The model predicts significant depletion of cellular calcium in response to agonist stimulation.

Observations on the Cell Biology of Tissue Factor in Endothelial Cells

1990 ◽  
Vol 63 (02) ◽  
pp. 298-302 ◽  
Author(s):  
Kiyoshi Andoh ◽  
Kjell Sverre Pettersen ◽  
Christiane Filion -Myklebust ◽  
Hans Prydz
Keyword(s):  
Endothelial Cells ◽  
Protein Synthesis ◽  
Plasma Membrane ◽  
Tissue Factor ◽  
Cell Biology ◽  
Umbilical Vein ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

SummaryHuman umbilical vein endothelial cells (HUVEC) are inducible for tissue factor (TF) activity in culture. Based on experiments using ECGF (4–20 µg/ml) with heparin (90 µg/ml), we obtained the followingresults: 1) In confluent HUVEC cultures, ECGF had essentially no influence on the levels of inducible TF. 2) In growing HUVEC cultures, ECGF reduced the TF response shortly after seeding but full response was regained when cells were kept confluent for 2–3 days. 3) Although secondary cultures responded best to TF induction in the absence of ECGF, the response was essentially equal over at least 8 passages in the prcgcncc of ECGF 1) Of total cellular TF induced in HUVEC, about 25% was available on the surface, and less than 4% was released with theshed plasma membrane vesicles. The proportion of total TF a>ctivity available on the surface of mtact cells wasnot influenced by the presence of ECGF 5) T½, for the decay of TF activity induced was 8.3—9.5 h, whereas in HUVEC when protein synthesis was blocked afterIF induction a T½ of about 30 h was found.

scholarly journals Monomeric C-Reactive Protein Decreases Acetylated LDL Uptake in Human Endothelial Cells

2009 ◽  
Vol 55 (9) ◽  
pp. 1728-1731 ◽  
Author(s):  
Susanne B Schwedler ◽  
Thomas Hansen-Hagge ◽  
Matthias Reichert ◽  
Daniel Schmiedeke ◽  
Reinhard Schneider ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Endothelial Cells ◽  
Umbilical Vein ◽  
Oxidized Ldl ◽  
Human Endothelial Cells ◽  
Rt Pcr ◽  
C Reactive Protein ◽  
Human Umbilical Vein ◽  
Reactive Protein ◽  
Umbilical Vein Endothelial Cells

Abstract Background: C-reactive protein (CRP) is a risk marker for cardiovascular disease and has been implicated in atherogenesis. In atherosclerotic plaques, it colocalizes with oxidized LDL (oxLDL) and promotes oxLDL uptake by macrophages, suggesting an important cross-talk between CRP and lipid processing. A growing body of evidence indicates the existence of distinct configurations of human CRP, native pentameric (nCRP) and structurally modified monomeric (mCRP), that elicit opposing bioactivities in vitro and in vivo. Here, we tested the impact of mCRP and nCRP on the uptake of acetylated LDL (acLDL), which is internalized by receptors similar to those of oxLDL in human endothelial cells. Methods: We cultured human umbilical vein endothelial cells (HUVECs) for 8 h with mCRP or nCRP (10–100 mg/L) and measured the uptake of acLDL (10–100 mg/L) over a 20-h period by FACS analysis. To assess the receptors involved, we used function-blocking antibodies against Fc γ receptor CD16 (FcγRIII) and CD32 (FcγRII), and RT-PCR analysis of CD16, CD32, and the receptor for oxidized LDL (LOX-1). Uptake of acLDL and CRP isoforms was visualized by immunofluorescence. Results: Culture of HUVECs with mCRP, but not nCRP, decreased uptake of acLDL, which was not prevented by anti-CD16 or anti-CD32 antibodies. LOX-1, CD16, and CD32 were undetectable by RT-PCR. Immunofluorescence showed decreased cytoplasmic acLDL staining in human umbilical vein endothelial cells (HUVECs) treated with mCRP, but not with nCRP. Conclusions: Monomeric CRP, but not nCRP, decreased acLDL uptake in human endothelial cells independent of CD16, CD32, or LOX-1. Our data support a protective role of mCRP in cardiovascular disease.

scholarly journals Synthesis and secretion of thrombospondin by cultured human endothelial cells.

1982 ◽  
Vol 93 (2) ◽  
pp. 343-348 ◽  
Author(s):  
D F Mosher ◽  
M J Doyle ◽  
E A Jaffe
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Human Platelet ◽  
Umbilical Vein ◽  
Rabbit Antiserum ◽  
Two Dimensional ◽  
Human Endothelial Cells ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

Thrombospondin, the major glycoprotein released from alpha-granules of thrombin-stimulated platelets, is a disulfide-bonded trimer of 160 kilodalton subunits and apparently functions as a platelet lectin. Because cultured human umbilical vein endothelial cells synthesize and secrete a glycoprotein (GP-160) which is a disulfide-bonded multimer of 160 kdalton subunits, the possibility that GP-160 is thrombospondin was investigated. Tritiated GP-160 could be immunoisolated from [3H]leucine-labeled endothelial cell postculture medium using a rabbit antiserum to human platelet thrombospondin. Thrombospondin and GP-160 comigrated in two different two-dimensional electrophoretic systems. Both proteins are disulfide-bonded trimers of acidic 160-kdalton subunits. A competitive radioimmunoassay for binding of 125I-thrombospondin to the rabbit antibodies indicated that 49 micrograms of thrombospondin antigen per 10(6) confluent endothelial cells accumulated in postculture medium over 24 h. Thus, endothelial cells secrete large amounts of a glycoprotein that is identical or very similar to platelet thrombospondin.

T-cadherin is located in the nucleus and centrosomes in endothelial cells

2009 ◽  
Vol 297 (5) ◽  
pp. C1168-C1177 ◽  
Author(s):  
Alexandra V. Andreeva ◽  
Mikhail A. Kutuzov ◽  
Vsevolod A. Tkachuk ◽  
Tatyana A. Voyno-Yasenetskaya
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Umbilical Vein ◽  
Subcellular Fractionation ◽  
Considerable Proportion ◽  
Human Endothelial Cells ◽  
Multinuclear Cells ◽  
Umbilical Vein Endothelial Cells ◽  
Interfering Rna ◽  
Intracellular Domains

T-cadherin (H-cadherin, cadherin 13) is upregulated in vascular proliferative disorders and in tumor-associated neovascularization and is deregulated in many cancers. Unlike canonical cadherins, it lacks transmembrane and intracellular domains and is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. T-cadherin is thought to function in signaling rather than as an adhesion molecule. Some interactive partners of T-cadherin at the plasma membrane have recently been identified. We examined T-cadherin location in human endothelial cells using confocal microscopy and subcellular fractionation. We found that a considerable proportion of T-cadherin is located in the nucleus and in the centrosomes. T-cadherin colocalized with a centrosomal marker γ-tubulin uniformly throughout the cell cycle at least in human umbilical vein endothelial cells. In the telophase, T-cadherin transiently concentrated in the midbody and was apparently degraded. Its overexpression resulted in an increase in the number of multinuclear cells, whereas its downregulation by small interfering RNA led to an increase in the number of cells with multiple centrosomes. These findings indicate that deregulation of T-cadherin in endothelial cells may lead to disturbances in cytokinesis or centrosomal replication.

Serum from healthy pregnant women reduces oxidative stress in human umbilical vein endothelial cells

2002 ◽  
Vol 103 (1) ◽  
pp. 53-57 ◽  
Author(s):  
Fortunato SCALERA ◽  
Tina FISCHER ◽  
Dietmar SCHLEMBACH ◽  
Ernst BEINDER
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Pregnant Women ◽  
Reduced Glutathione ◽  
Umbilical Vein ◽  
Lipid Peroxides ◽  
Human Endothelial Cells ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

This study was conducted to compare the effects of serum from healthy pregnant women and that from pregnant women with pre-eclampsia on oxidative stress in endothelial cells in culture. Human umbilical vein endothelial cells (HUVECs) were incubated with serum from 18 pre-eclamptic, 18 healthy pregnant and 18 healthy non-pregnant women for 24h. The levels of reduced glutathione (GSH) and lipid peroxides (LPOs) were measured in endothelial cell lysates. Measurement of malondialdehyde in combination with 4-hydroxyalkenals has been used as an indicator of LPOs. Serum from healthy pregnant women decreased significantly the LPO content in HUVECs in comparison with serum from pre-eclamptic women and healthy non-pregnant women (30.7±6.6 compared with 39.3±10.9 and 41.0±12.7pmol/mg of protein respectively; P<0.003 and P<0.01 respectively). No differences in GSH content between the three groups (18.3±2.1nmol/mg of protein for healthy pregnant, 19.2±3.3nmol/mg for pre-eclamptic and 18.3±2.0nmol/mg for healthy non-pregnant women) were found. Thus serum from normal pregnant women contains a factor(s) that decreases oxidative stress in human endothelial cells. This mechanism might be altered in pre-eclampsia.

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