Plateiet-Activating Factor (1-0-Alkyl-2-Acetyl-sn-Glyceryl-3-Phosphorycholine) Stimulates Prostacyclin Synihesis By Cultured Human Endothelial Cells

l-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (Ac-GEPC) has recently been established to possess biological properties identical to those of platelet-activating factor (PAF) released from stimulated mast cells and leucocytes (Demopoulos et al, J. Biol. Chem. 254:9355, 1979). Shaw et al (J. Imuunol. 121:1939, 1978) reported that PAF is capable of stimulating platelet thranbaxane A2 synthesis. We examine here whether synthetic Ac-GEPC stimulates PGI2 synthesis in human endothelial cell cultures. Human umbilical vein endothelial cells during the third passage were grown to confluency in 35 x 10 ran dishes and challenged with Na arachidonate(NaA), 8 x 10-5M or Ac-GEPC, 5 x 10-% (a concentration which produced optimal platelet aggregation in human platelet-rich plasma). PGI2 production was monitored through radioimmunoassays of its metabolite, 6-keto-PGF1α. After washing endothelial cell layers twice, the agonists were added in tris-Tyrodes buffer, pH 7.2 containing 2.5 g/L bovine serum albumin. Representative timed experiments produced the following results expressed in pM 6-keto-PGF1α/105 cells for NaA and Ac-GEPC respectively after the noted times of incubation: 10s: 274/467, 20s: 630/1136, 30s: 1111/1005, 60s: 1757/1217, 90s: 1842/1272, 120s: 1115/698, 180s: 1188/453. These data indicate that Ac-GEPC stimulates the production of PGI2 at a rate roughly comparable to that achieved by stimulation with NaA, reaching a maximum at 60 and 90s in these experiments. The decrease, compared to peak levels, in iranunoreactive 6-keto-PGF1α at 120 and 180s of 23% and 36% for NaA stimulated cells and 45% and 65% for Ac-GEPC stimulated cells suggests that enzymes are activated during the experiment which cause conversion of PGI2 into metabolites other than 6-keto-PGF1α (e.g. 6-keto-PGE1). These results suggest another theoretically important negative feedback mechanism for a potent platelet proaggregatorv substance.

Abstract P069: Microvesicles From Enos-suppressed Endothelial Cells Induce Endothelial Cell Dysfunction

Hypertension ◽

10.1161/hyp.76.suppl_1.p069 ◽

2020 ◽

Vol 76 (Suppl_1) ◽

Author(s):

Vinicius P Garcia ◽

Jamie G Hijmans ◽

Kelly A Stockelman ◽

Madden Brewster ◽

Hannah Fandl ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Umbilical Vein ◽

No Production ◽

Cell Dysfunction ◽

Enos Activity ◽

And Function ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Introduction: Endothelial nitric oxide synthase (eNOS) activity is critical to vascular health. Impaired eNOS activity and diminished NO production are common characteristics of a proatherogenic, dysfunctional endothelial phenotype that is associated with cardiovascular risk factors and disease. Extracellular microvesicles, particularly endothelial cell derived microvesicles (EMVs) represent novel mechanistic mediators of endothelial dysfunction and vascular disease. It is unknown whether eNOS suppression affects EMV number and function. We tested the following hypotheses: 1) eNOS blockade increases EMV release; and 2) EMVs derived from eNOS-suppressed cells adversely affect endothelial cell inflammation, apoptosis and NO production. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with the eNOS inhibitor, L-N G -Nitroarginine methyl ester (L-NAME; 300mM) for 24 h. EMVs (CD144 + ) released into the supernatant from cells treated with L-NAME or vehicle were isolated and quantified by flow cytometry. Fresh HUVECs were then treated with either L-NAME-derived or control EMVs for 24 h. To evaluate the role of endocytosis on the endothelial effects of EMVs, HUVECs were pre-incubated (12 h) with EIPA, filipin and chlorpromazine for 2 h, and all experiments repeated. Results: EMV release was markedly higher (~100%; P<0.05) in cells treated with L-NAME compared with control (81±6 vs. 40±7 EMV/μL). L-NAME-generated EMVs induced significantly higher release of IL-6 (38.4±5.1 vs. 21.0±1.9 pg/mL) and IL-8 (38.9±3.5 vs. 27.2±3.1 pg/mL) as well as greater active NF-κB p65 (Ser-536) (9.7±0.7 vs. 6.1±0.6 AU) expression than control EMVs. The expression of activated-caspase-3 was significantly higher in the cells treated with L-NAME (9.5±1.1 vs. 6.4±0.4 AU). Total eNOS (97.1±8.2 vs. 157.5±15.6 AU), activated eNOS (4.9±1.2 vs. 9.1±1.3 AU) and NO production (5.0±0.8 vs. 7.0±0.6 μmol/L) were significantly lower in endothelial cells treated with EMVs from eNOS suppressed cells. Endocytosis blockers mitigated the deleterious endothelial effects of EMVs. Conclusion: eNOS-suppression increases EMV release. Moreover, EMVs from eNOS-suppressed cells increase endothelial cell inflammation and apoptosis and decrease NO production.

Thrombin-induced gap formation in confluent endothelial cell monolayers in vitro

Blood ◽

10.1182/blood.v62.3.549.549 ◽

1983 ◽

Vol 62 (3) ◽

pp. 549-556 ◽

Cited By ~ 109

Author(s):

M Laposata ◽

DK Dovnarsky ◽

HS Shin

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Umbilical Vein ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Gap Formation ◽

Culture Dish ◽

Cell Monolayers ◽

Abstract When thrombin is incubated with confluent monolayers of human umbilical vein endothelial cells in vitro, there is a change in the shape of the endothelial cells that results in gaps in the monolayer, disrupting the integrity of the endothelium and exposing the subendothelium. Using a grid assay to measure this phenomenon, we observed that up to 80% of the surface area once covered by cells was uncovered after a 15-min incubation with 10(-2) U/ml (10(-10)M) thrombin. The effect was apparent within 2 min and did not remove cells from the surface of the culture dish. The gaps in the monolayer completely disappeared within 2 hr after exposure to thrombin. The effect of thrombin was inhibited by preincubation of thrombin with hirudin or antithrombin III plus heparin or by preincubation of the monolayers with dibutyryl cyclic adenosine monophosphate (dbcAMP). Histamine also induced gap formation in endothelial cell monolayers. Both pyrilamine and cimetidine prevented the histamine-induced effect, but they had no effect on thrombin- induced gap formation. Intact monolayers were not disrupted by bradykinin, serotonin, C5a, or C3a. Our results suggest that small amounts of thrombin can induce repeated and transient exposure of the subendothelium, a situation believed to be conducive to atherogenesis and thrombosis.

Omentin-1 Ameliorated Free Fatty Acid-Induced Impairment in Proliferation, Migration, and Inflammatory States of HUVECs

Cardiology Research and Practice ◽

10.1155/2020/3054379 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9

Author(s):

Yubin Chen ◽

Fen Liu ◽

Fei Han ◽

Lizhi Lv ◽

Can-e Tang ◽

...

Keyword(s):

Endothelial Cells ◽

Fatty Acid ◽

Endothelial Cell ◽

Free Fatty Acid ◽

Cell Injury ◽

Umbilical Vein ◽

Quantitative Polymerase Chain Reaction ◽

Cell Counting ◽

Endothelial Cell Injury ◽

Objectives. Endothelial cell injury is a critical pathological change during the development of atherosclerosis. Here, we explored the effect of omentin-1 on free fatty acid- (FFA-) induced endothelial cell injury. Methods. An FFA-induced endothelial cell injury model was established to investigate the role of omentin-1 in this process. Cell proliferation was analyzed with the Cell Counting Kit assay and flow cytometry. Scratch and transwell assays were used to evaluate cell migration. Factors secreted by endothelial cells after injury were detected by western blotting, reverse-transcription quantitative polymerase chain reaction, and cellular fluorescence assay. Results. Omentin-1 rescued the FFA-induced impaired proliferation and migration capabilities of human umbilical vein endothelial cells (HUVECs). It decreased the number of THP-1 cells attached to HUVECs in response to injury and inhibited the FFA-induced proinflammatory state of HUVECs. Conclusion. Omentin-1 could partly ameliorate FFA-induced endothelial cell injury.

Endothelialized silicone aneurysm models for in vitro evaluation of flow diverters

Journal of NeuroInterventional Surgery ◽

10.1136/neurintsurg-2020-016859 ◽

2020 ◽

pp. neurintsurg-2020-016859

Author(s):

Alyssa McCulloch ◽

Ashley Turcott ◽

Gabriella Graham ◽

Sergey Frenklakh ◽

Kristen O'Halloran Cardinal

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Umbilical Vein ◽

Flow Diverter ◽

In Vitro Models ◽

Cell Coverage ◽

Platelet Endothelial Cell Adhesion ◽

Over Time

ObjectiveThe goal of this work was to endothelialize silicone aneurysm tubes for use as in vitro models for evaluating endothelial cell interactions with neurovascular devices. The first objective was to establish consistent and confluent endothelial cell linings and to evaluate the silicone vessels over time. The second objective was to use these silicone vessels for flow diverter implantation and assessment.MethodsSilicone aneurysm tubes were coated with fibronectin and placed into individual bioreactor systems. Human umbilical vein endothelial cells were deposited within tubes to create silicone vessels, then cultivated on a peristaltic pump and harvested at 2, 5, 7, or 10 days to evaluate the endothelial cell lining. A subset of silicone aneurysm vessels was used for flow diverter implantation, and evaluated for cell coverage over device struts at 3 or 7 days after deployment.ResultsSilicone vessels maintained confluent, PECAM-1 (platelet endothelial cell adhesion molecule 1) positive endothelial cell linings over time. These vessels facilitated and withstood flow diverter implantation, with robust cell linings disclosed after device deployment. Additionally, the endothelial cells responded to implanted devices through coverage of the flow diverter struts with increased cell coverage over the aneurysm seen at 7 days after deployment as compared with 3 days.ConclusionsSilicone aneurysm models can be endothelialized and successfully maintained in vitro over time. Furthermore, these silicone vessels can be used for flow diverter implantation and assessment.

Exogenous xanthine promotes neutrophil adherence to cultured endothelial cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1997.273.2.g342 ◽

1997 ◽

Vol 273 (2) ◽

pp. G342-G347

Author(s):

H. Ichikawa ◽

R. E. Wolf ◽

T. Y. Aw ◽

N. Ohno ◽

L. Coe ◽

...

Keyword(s):

Endothelial Cells ◽

Tissue Injury ◽

Umbilical Vein ◽

Platelet Activating Factor ◽

Adhesion Assay ◽

Neutrophil Adherence ◽

Paf Receptor ◽

Adhesive Effect ◽

Paf Receptor Antagonist

Oxidants generated by endothelial xanthine oxidase (XO) can help trigger free radical-mediated tissue injury. An important event in oxidant-mediated tissue injury is neutrophil-endothelial adhesion. Although activation of endothelial XO increases adhesion, little is known about xanthine in the adhesive effect of XO. This study examined administered xanthine on the adhesion of neutrophils. Endothelial [human umbilical vein endothelial cells (HUVEC)] monolayers were exposed to xanthine (15 min), and neutrophils were allowed to adhere to HUVEC in an adhesion assay. Adhesion was dose dependently increased by xanthine (3-100 microM). Either catalase (1,000 U/ml), oxypurinol (XO inhibitor; 100 microM), or platelet-activating factor (PAF) receptor antagonist (WEB 2086; 10 microM) reduced neutrophil adhesion. Superoxide dismutase (1,000 U/ml) had no effect. Pretreatment of HUVEC with 50 microM tungsten also blocked xanthine-induced adherence. Adhesion was also inhibited by preincubation with 100 U/ml heparin. Finally, anti-P-selectin antibody (PB1.3; 20 micrograms/ml) attenuated adhesion. Our results indicate that xanthine may promote neutrophil-endothelial adhesion via a hydrogen peroxide- and PAF-mediated P-selectin expression.

Modulation Of PGI2-Like Activity Of Human Endothelial Cells By An Extracellular Collagen Matrix

10.1055/s-0038-1653228 ◽

1981 ◽

Author(s):

K M Spiegel ◽

S F Mohammad ◽

H Y K Chuang ◽

R G Mason

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Umbilical Vein ◽

Collagen Matrix ◽

Significant Loss ◽

Experimental Conditions ◽

Cell Lysates ◽

Collagen Coating ◽

Human umbilical vein endothelial cells were grown on several artificial matrices including collagen and on unmodified plastic dishes. Freeze thaw preparations of confluent monolayer endothelial cell cultures were assayed for PGI2-like activity by testing for inhibition of platelet a88regation. The cells grown on a collagen matrix expressed slightly less PGI2-like activity initially compared to the cells grown on plastic. When the PGI2like activity of the cell lysates was examined over a period of three hours after the initial preparation, endothelial cells grown on a collagen matrix exhibited a more rapid loss of activity. Preliminary quantitative evaluations suggest that lysates derived from cells grown on unmodified plastic retained 75% PGI2 like activity at 15 min, at which point collagen grown cells exhibited essentially no PGI2-like activity. Furthermore, as shown in the figure, under identical experimental conditions, cell lysates obtained from endothelial cells grown on unmodified plastic continued to express approximately 50% of the initial PGI2-like activity after one hour. Since addition of purified PGI2 or cell lysates in vitro to the collagen coating used as tissue culture substrate failed to cause any significant loss of platelet aggregation inhibitory activity beyond the normal rate of decay of PGI2 it appears likely that the reduction of PGI2-like activity may be associated with changes in the substrate collagen, possibly effected by the endothelial cell layer. Alternatively, the reduction of activity may be related to differences in the endothelial cells caused by growth on the collagen substrate.

MODULATION of PROSTACYCLIN PRODUCTION BY HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS WITH ECGF/HEPARIN MEDIUM : ROLE OF CELLULAR DENSITY AT CONFLUENCE

10.1055/s-0038-1643376 ◽

1987 ◽

Author(s):

O BOUTHERIN-FALSON ◽

N BLAES

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Growth Factor ◽

Umbilical Vein ◽

Calf Serum ◽

Lower Amount ◽

Endothelial Cell Proliferation ◽

Human Umbilical Vein ◽

Prostacyclin (PGI2) is a major product of arachidonic acid metabolism in vascular endothelial cells. In addition to the role of exogenous agents, its production could be modulated by culture conditions : proliferative state, medium renewal, subcultivation... The use of endothelial cell growth factor (ECGF) associated with heparin has been shown to improve human endothelial cell proliferation. Here we report that human umbilical vein endothelial cells (HUVEC) grown in that medium produce less prostacyclin than without growth factor.HUVEC were cultured in RPMI-199 1:1 + 20% fetal calf serum, added or not with ECGF (Bovine hypothalamus extract BTI Cambridge, 24 ug/ml) and heparin (from porcine intestinal mucosa, Signa, 90 ug/ml). After 4 days in culture, medium was removed and replaced by Tyrode Hepes buffer and basal production was measured after 20 min. Cells were then submitted to 5 min thrombin to assess PGI2 production in stimulated conditions. PGI2 production was estimated by specific radioimmunoassay for 6 keto PGFjalpha. For each point, cell number in the culture was counted after Trypsin EDTA treatment. In the present study, cells grown in ECGF-heparin medium produce lower amount of PGI2, compared to heparin or control medium. This result was observed in both basal and stimulated conditions. For each medium (ECGF-heparin, heparin, control), correlations between PGI2 production per cell and log cell density were shown to be significantly negative.These observations suggest that ECGF effect on PGI2 production could be a consequence of its growth factor activity, notably by the fact that it leads to an endothelial monolayer made of more numerous cells. Since it is now suggested by a number of clinical observations that PGI2 is rather produced in pathological conditions, culture models showing a weak production of PGI2 appear in that connection doser to the physiological conditions.

Up-regulation of the Notch ligand Delta-like 4 inhibits VEGF-induced endothelial cell function

Blood ◽

10.1182/blood-2005-03-1000 ◽

2006 ◽

Vol 107 (3) ◽

pp. 931-939 ◽

Cited By ~ 245

Author(s):

Cassin Kimmel Williams ◽

Ji-Liang Li ◽

Matilde Murga ◽

Adrian L. Harris ◽

Giovanna Tosato

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Cell Function ◽

Umbilical Vein ◽

Endothelial Cell Proliferation ◽

Vegf Receptor ◽

Endothelial Cell Function ◽

Notch Ligand ◽

Neuropilin 1 ◽

AbstractDelta-like 4 (Dll4), a membrane-bound ligand for Notch1 and Notch4, is selectively expressed in the developing endothelium and in some tumor endothelium, and it is induced by vascular endothelial growth factor (VEGF)-A and hypoxia. Gene targeting studies have shown that Dll4 is required for normal embryonic vascular remodeling, but the mechanisms underlying Dll4 regulatory functions are currently not defined. In this study, we generated primary human endothelial cells that overexpress Dll4 protein to study Dll4 function and mechanism of action. Human umbilical vein endothelial cells retrovirally transduced with Dll4 displayed reduced proliferative and migratory responses selectively to VEGF-A. Expression of VEGF receptor-2, the principal signaling receptor for VEGF-A in endothelial cells, and coreceptor neuropilin-1 was significantly decreased in Dll4-transduced endothelial cells. Consistent with Dll4 signaling through Notch, expression of HEY2, one of the transcription factors that mediates Notch function, was significantly induced in Dll4-overexpressing endothelial cells. The γ-secretase inhibitor L-685458 significantly reconstituted endothelial cell proliferation inhibited by immobilized extracellular Dll4 and reconstituted VEGFR2 expression in Dll4-overerexpressing endothelial cells. These results identify the Notch ligand Dll4 as a selective inhibitor of VEGF-A biologic activities down-regulating 2 VEGF receptors expressed on endothelial cells and raise the possibility that Dll4 may be exploited therapeutically to modulate angiogenesis.

Thrombopoietin Stimulates the Formation of Endothelial Cell-Cell Contacts and Activates the Small GTPase Rap1.

Blood ◽

10.1182/blood.v110.11.3713.3713 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3713-3713

Author(s):

Marloes R. Tijssen ◽

Franca di Summa ◽

Anne-Marieke van Stalborch ◽

C. Ellen Van der Schoot ◽

Jaap D. van Buul ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Electrical Resistance ◽

Umbilical Vein ◽

Small Gtpase ◽

Endothelial Monolayer ◽

Impedance Sensing ◽

Cell Contacts ◽

Cell Cell

Abstract In addition to hematopoietic cells, the receptor for thrombopoietin (Tpo), c-Mpl, is expressed on human umbilical vein endothelial cells (HUVEC). In this study, we demonstrated that Mpl mRNA and protein can also be detected in immortalized human bone marrow endothelial cells (HBMEC). Furthermore, it was investigated whether Tpo affects endothelial monolayer integrity by either cell spreading or cell-cell contacts. By continuously monitoring the electrical resistance of freshly plated HUVEC or HBMEC with ECIS (electric cell-substrate impedance sensing), we observed a significant increase in the electrical resistance when endothelial cells were treated with 100 ng/ml of Tpo just prior to seeding, as shown in the figure below. This increase in electrical resistance was not due to increased cell adhesion or proliferation of the endothelial cells. However, a significant increase in initial spreading could be detected. Besides its effect on spreading, Tpo increased the electrical resistance of a confluent endothelial monolayer when compared to untreated monolayers. This indicates that Tpo promotes the formation of endothelial cell-cell contacts. Indeed, a decrease in immunostaining levels of phosphotyrosine could be observed in monolayers treated with Tpo. Also the junctional protein VE-cadherin showed diminished co-localization with phosphotyrosine staining when cells were treated with Tpo. The small GTPase Rap1 plays an important role in the formation of endothelial cell-cell contacts. In a pull-down experiment, using RalGDS as bait for active Rap1, we could demonstrate that Tpo transiently activates Rap1 in HUVEC. Thus, these results may put Tpo forward as a regulator of endothelial cell-cell contacts, possibly after vascular damage and subsequent release of Tpo by activated platelets. To our knowledge, Tpo is the first cytokine exerting this effect on endothelial cells. Figure Figure