Effects of experimental conditions on the production of interleukin-1α and -1β by human endothelial cells cultured in vitro

SummaryIn most studies showing cardio- and vasculoprotective effects of estrogens, 17β-estradiol was used and little information on possible effects of different estrogen metabolites is yet available. We investigated whether particular estrogen metabolites are effective in counteracting inflammatory activation of human endothelium. Human endothelial cells were incubated with 17α-dihydroequilenin, 17β-dihydroequilenin, δ-8,9-dehydroestrone, estrone and 17β-estradiol and stimulated with interleukin (IL)-1α.The expression of IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) was determined. 17β-dihydroequilenin and 17β-estradiol at a concentration of 1µM reduced IL-1α-induced up regulation of IL-6, IL-8 and MCP-1 close to control levels. When both compounds were used in combination an additive effect was observed. 17α-dihydroequilenin and δ-8,9-dehydroestrone showed a similar anti-inflammatory effect only when used at 10µM whereas estrone had no effect. The effect of 17β-dihydroequilenin on IL-1α-induced production of IL-6, IL-8 and MCP-1 was reversed by the estrogen receptor antagonist ICI 182,780. 17β-dihydroequilenin also inhibited IL-1α-induced translocation of p50 and p65 to the nucleus of the cells. We have identified the estrogen metabolite 17β-dihydroequilenin, as an inhibitor of inflammatory activation of human endothelial cells. Characterization of specific estrogens – as shown in our study – could provide the basis for tailored therapies, which might be able to achieve vasoprotection without adverse side effects.

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An In Vitro Toxicity Assay for Human Endothelial Cells

Alternatives to Laboratory Animals ◽

10.1177/026119298801600109 ◽

1988 ◽

Vol 16 (1) ◽

pp. 38-41

Author(s):

Rosella Sbarbati ◽

Maria Luisa Schinetti ◽

Maria Scarlattini

Keyword(s):

Oxidative Stress ◽

Endothelial Cells ◽

Umbilical Vein ◽

Vascular Diseases ◽

In Vitro Toxicity ◽

Human Endothelial Cells ◽

Experimental Conditions ◽

Umbilical Vein Endothelial Cells

Cultured human endothelial cells can replace living animals in studying the toxic role of noxious agents in the pathogenesis of vascular diseases and in the elucidation of the mechanism of action of protective drugs. Preliminary data are presented which examine the effects that oxidative stress produces on human endothelial cells in vitro. Human umbilical vein endothelial cells were subjected to an anoxia-re-oxygenation treatment and tested for the production of Super Oxide Dismutase (SOD)-inhibitable superoxide radicals. The results show that under our experimental conditions endothelial cells produce oxygen-free radicals and that the generation reaches a maximum after an anoxic challenge of 20 minutes. We conclude that the in vitro system presented in this paper could be a suitable tool for further studies on the effects of oxidative stress on the vascular endothelium, which mimics the in vivo conditions of re-perfusion after heart ischemia.

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The biosynthesis of extracellular-matrix components by bovine retinal endothelial cells displaying distinctive morphological phenotypes

Biochemical Journal ◽

10.1042/bj2350375 ◽

1986 ◽

Vol 235 (2) ◽

pp. 375-383 ◽

Cited By ~ 27

Author(s):

A E Canfield ◽

A M Schor ◽

S L Schor ◽

M E Grant

Keyword(s):

Endothelial Cells ◽

Type I ◽

Collagen Gels ◽

Early Passage ◽

Experimental Conditions ◽

Retinal Endothelial Cells ◽

Type Iv ◽

Extracellular Matrix Components ◽

Cells Cultured

Previous studies have indicated that the morphology and behaviour of bovine retinal microvessel endothelial cells are influenced by culture conditions in vitro. Data are presented here concerning the biosynthesis of matrix macromolecules by bovine retinal endothelial cells cultured under conditions in which the cells display either the ‘cobblestone’ or the ‘sprouting’ phenotype. Newly synthesized matrix proteins were identified by their characteristic electrophoretic mobilities, immunoprecipitation with specific antibodies, susceptibilities to enzymic digestions and chromatographic behaviour. Type IV procollagen was the major collagenous species synthesized by early-passage cells forming a ‘cobblestone’ monolayer. In contrast, cells displaying the ‘sprouting’ morphology switched to the predominant synthesis of interstitial fibrillar collagens (types I and III). Fibronectin was synthesized by retinal endothelial cells under all the experimental conditions studied. A non-collagenous glycoprotein of Mr approx. 47,000 was also a major biosynthetic product of these cells. The synthesis of thrombospondin was very much dependent on the nature of the substratum on which the cells were cultured. This glycoprotein was synthesized in large amounts by ‘cobblestone’ endothelial cells cultured on gelatin-coated dishes, whereas its synthesis was markedly decreased by culturing the cells on collagen gels, and the protein appeared to be absent when the cells were plated within collagen gels (‘sprouting’ cells). Late-passage retinal cells synthesized predominantly type I procollagen, variable amounts of type III procollagen and only traces of type IV procollagen, irrespective of whether the cells displayed a ‘cobblestone’ or ‘sprouting’ morphology.

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Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656088 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0975-0980 ◽

Cited By ~ 23

Author(s):

Angel Gálvez ◽

Goretti Gómez-Ortiz ◽

Maribel Díaz-Ricart ◽

Ginés Escolar ◽

Rogelio González-Sarmiento ◽

...

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Tissue Factor ◽

Platelet Adhesion ◽

Umbilical Vein ◽

Procoagulant Activity ◽

Experimental Conditions ◽

Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

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Acute damage to human endothelial cells by brief exposure to contrast media in vitro.

Radiology ◽

10.1148/radiology.147.3.6844604 ◽

1983 ◽

Vol 147 (3) ◽

pp. 681-684 ◽

Cited By ~ 50

Author(s):

F Laerum

Keyword(s):

Endothelial Cells ◽

Contrast Media ◽

Human Endothelial Cells

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Vascular repair utilising immobilised heparin conjugate for protection against early activation of inflammation and coagulation

Thrombosis and Haemostasis ◽

10.1160/th14-09-0724 ◽

2015 ◽

Vol 113 (06) ◽

pp. 1312-1322 ◽

Cited By ~ 18

Author(s):

Sofia Nordling ◽

Jaan Hong ◽

Karin Fromell ◽

Fredrik Edin ◽

Johan Brännström ◽

...

Keyword(s):

Endothelial Cells ◽

Thrombus Formation ◽

Innate Immune ◽

Human Endothelial Cells ◽

Coagulation Activation ◽

Ischaemia Reperfusion Injury ◽

Novel Approach ◽

One Step ◽

Chamber Model

SummaryIschaemia-reperfusion injury (IRI) poses a major challenge in many thrombotic conditions and in whole organ transplantation. Activation of the endothelial cells and shedding of the protective vascular glycocalyx during IRI increase the risk of innate immune activation, cell infiltration and severe thrombus formation, promoting damage to the tissue. Here, we present a novel one-step strategy to protect the vasculature by immobilisation of a unique multi-arm heparin conjugate to the endothelium. Applying a new in vitro blood endothelial cell chamber model, the heparin conjugate was found to bind not only to primary human endothelial cells but also directly to the collagen to which the cells adhered. Incubation of hypoxic endothelial cells with freshly drawn human blood in the blood chambers elicited coagulation activation reflected by thrombin anti-thrombin formation and binding of platelets and neutrophils. Immobilisation of the heparin conjugate to the hypoxic endothelial cells created a protective coating, leading to a significant reduction of the recruitment of blood cells and coagulation activation compared to untreated hypoxic endothelial cells. This novel approach of immobilising multi-arm heparin conjugates on the endothelial cells and collagen of the basement membrane ensures to protect the endothelium against IRI in thrombotic disorders and in transplantation.

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PGI2 Production In Human Endothelial Cells Cultured In Diabetic And Nondiabetic Serum

10.1055/s-0038-1652118 ◽

1981 ◽

Author(s):

R C Paton ◽

R Guillot ◽

Ph Passa

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Vascular Complications ◽

Proliferative Retinopathy ◽

Vascular Endothelial ◽

Human Endothelial Cells ◽

Bovine Thrombin ◽

Control Serum ◽

Umbilical Cords ◽

Cells Cultured

Reduced levels of prostaglandin I2 (PGI2) may contribute to the platelet hyper-reactivity and vascular complications found in diabetes mellitus. This study compared PGI2 production (PGI2-like activity and 6-keto-PGF1α levels) by vascular endothelial cells cultured in the presence of serum from 15 diabetics with proliferative retinopathy (5 treated by surgical hypophysectomy) and 15 sex-matched nondiabetic controls. Endothelial cells from human umbilical veins were cultured in M199 with either 20 % diabetic or control serum. At confluence, cultures were washed and stimulated with 0.1 NIH u/ml bovine thrombin. After 2 min incubation, the supernatant was tested for i)PGI2-like activity on ADP- induced platelet aggregation, results expressed as % inhibition and ii) 6-keto-PGF1α by radioimmunoassay, results expressed as nmol/ml. There was a significant correlation between PGI2-like activity and 6-keto-PGF-1α levels (r 0.78, p<0.001). The liberation of PGI2 from endothelial cells from different umbilical cords varied, but both PGI2-like activity (mean± SEM 21.9± 4.8 vs 28.3± 5.1 p<0.05) and 6-keto-PGF-1α (3.15± 0.68 vs 3.95 ±0.91 nmol/ml, p <0.05)were significantly lower in superantant from cells cultured in the presence of diabetic compared to control serum. PGI2 production was not significantly different in cells cultured with serum from hypophysectomised and nonhypophysectomised diabetics.These results suggest that serum from diabetics with proliferative retinopathy contains factors which impair the release or production of PGI2 by endothelial cells and that this effect is not mediated by the pituitary.

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