scholarly journals Differential regulation of von Willebrand factor exocytosis and prostacyclin synthesis in electropermeabilized endothelial cell monolayers

1995 ◽  
Vol 309 (2) ◽  
pp. 473-479 ◽  
Author(s):  
J A Frearson ◽  
P Harrison ◽  
M C Scrutton ◽  
J D Pearson
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Secretory Granules ◽  
Guanine Nucleotide Binding Protein ◽  
Intact Cells ◽  
Nucleoside Triphosphates ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Prostacyclin Synthesis

We have developed a system to permeabilize human umbilical vein endothelial cells in monolayer culture by application of a high-voltage electric field. The permeabilized preparation allows access of small molecules (M(r) < 1000) without loss of large cytosolic proteins. Electropermeabilized cells exocytose highly multimeric von Willebrand factor from secretory granules in response to added Ca2+ (EC50 = 0.8 +/- 0.02 microM), with levels comparable with those observed on stimulation of intact endothelial cells by physiological agonists. MgATP2- potentiates Ca(2+)-driven von Willebrand factor secretion. Other nucleoside triphosphates, but not non-hydrolysable analogues, can replace ATP. Electropermeabilized cells also synthesize and release prostacyclin in response to added Ca2+ (EC50 = 0.3 +/- 0.08 microM), but nucleoside triphosphates markedly inhibit, whereas nonhydrolysable GTP analogues increase, Ca(2+)-driven prostacyclin synthesis. We conclude that elevation of the intracellular [Ca2+] is sufficient to cause efficient exocytosis of von Willebrand factor from permeabilized cells, despite evidence that additional second messengers are needed in intact cells. We find no evidence in endothelial cells for a guanine nucleotide-binding protein promoting exocytosis, although one is clearly involved in stimulating Ca(2+)-driven prostacyclin synthesis.

Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

1997 ◽  
Vol 77 (06) ◽  
pp. 1182-1188 ◽  
Author(s):  
Ulrich M Vischer ◽  
Claes B Wollheinn
Keyword(s):  
Endothelial Cells ◽  
Adenylate Cyclase ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Free Calcium ◽  
Camp Content ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

Hydrogen peroxide stimulates exocytosis of von Willebrand factor in human umbilical vein endothelial cells

2017 ◽  
Vol 44 (5) ◽  
pp. 531-537 ◽  
Author(s):  
P. V. Avdonin ◽  
A. A. Tsitrina ◽  
G. Y. Mironova ◽  
P. P. Avdonin ◽  
I. L. Zharkikh ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract 37: Secretion of von Willebrand Factor by Endothelial Cells Links Salt to Hypercoagulability and Thrombosis

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Natalia I Dmitrieva ◽  
Maurice B Burg
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Salt Intake ◽  
Umbilical Vein ◽  
Coagulation Cascade ◽  
Water Restriction ◽  
Fibrinogen Degradation Product ◽  
Modifiable Factors ◽  
Von Willebrand ◽  
Willebrand Factor

Hypercoagulability increases the risk of thrombi that cause cardiovascular events. Dehydration and hypernatremia are often accompanied by thrombosis, but the mechanisms are not clear. Von Willebrand Factor is secreted by endothelium, affecting aggregation of platelets and promoting activation of the coagulation cascade and formation of thrombi. Here we show that in culture of primary Human Umbilical Vein Endothelia Cells, elevating medium osmolality to 320-380 mosmol/kg by adding NaCl reversibly increases both vWF mRNA and vWF secretion. The high NaCl increases expression of tonicity regulated transcription factor NFAT5 and its binding to promoter of vWF gene, suggesting that vWF upregulation is caused by hypertonic signaling. To elevate NaCl in vivo, we modeled mild dehydration, subjecting mice to water restriction (WR) for 9 days by feeding them with gel food containing 30% of water. Such WR elevates blood sodium from 145.1±0.5 to 150.2±1.3 mmol/l and activates hypertonic signaling as evidenced from increased expression of NFAT5 in tissues. WR increased vWF mRNA in liver and lung and raised vWF protein in blood. Immunostaining of liver revealed increased production of vWF protein by endothelium and increased number of microthrombi inside capillaries. WR also increased blood level of D-dimer, a fibrinogen degradation product indicative of ongoing coagulation and thrombolysis. We conclude that elevation of extracellular sodium within the physiological range raises expression and secretion of von Willebrand Factor sufficiently to increase coagulability of blood and risk of thrombosis. The results suggest that hydration and salt intake are modifiable factors that affect coagulability and thrombosis through high salt-dependent secretion of vWF from endothelial cells.

ABNORMAL EXPRESSION OF VON WILLEBRAND FACTOR BY ENDOTHELIAL CELLS FROM A PATIENT WITH TYPE IIA VON WILLEBRAND’ S DISEASE

1987 ◽  
Author(s):  
Richard B Levene ◽  
Francis M Booyse ◽  
Juan Chediak ◽  
Therodore S Zimmerman ◽  
David M Livingston ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Kinetic Studies ◽  
Protein Product ◽  
Polymerization Reaction ◽  
Full Spectrum ◽  
Endogenous Proteases ◽  
Von Willebrand ◽  
Willebrand Factor

Studies were conducted to characterize the biosynthesis of von Willebrand factor (vWf) by cultured endothelial cells (EC) derived from the umbilical vein of a patient with type HA von Willebrand’ s disease. The patient’ s EC, compared with those from normal individuals, produced vWf which had decreased amounts of large multimers and an increase in rapidly migrating satellite species, features which are characteristic of plasma vWf from patients with type IIA von Willebrand’ s disease. The typQ IIA EC produced a full spectrum of vWf multimers in both cell lysates and post-culture medium, although the relative amounts of the larger species were decreased. The large multimers were degraded in conjunction with the appearance of rapidly migrating satellites which contained =170 kDa proteolytic fragments. Kinetic studies demonstrated that the =170 kDa species is not a primary translation product Normal metabolically labeled vWf, incubated with either the patient’ s EC or medium conditioned by these cells, was not similarly degraded. These results demonstrated that this patient’ s clinical phenotype is due to abnormal proteolysis and not to a primary failure of subunit oligomerization. Moreover, the increased degradation is attributable to increased proteolytic sensitivity of an abnormal vWf molecule rather than to pathologically elevated levels of endogenous proteases. Experiments using monoclonal antibodies which recognize either N- or C-associated epitopes have localized the defect to the N-terminal portion of the vWf molecule, which is believed to be involved in the inter-dimer polymerization reaction. The type DA EC also contained a single vWf mRNA species which comigrated with that from normal EC. However, the type HA EC contained 8-10 fold more vWf mRNA than their normal counterparts. These results suggest that the functional defect in this patient is caused by a subtle mutation in the vWf coding sequence leading to increased proteolytic sensitivity of its protein product

scholarly journals Weibel-Palade body size modulates the adhesive activity of its von Willebrand Factor cargo in cultured endothelial cells

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Francesco Ferraro ◽  
Mafalda Lopes da Silva ◽  
William Grimes ◽  
Hwee Kuan Lee ◽  
Robin Ketteler ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Granules ◽  
Vascular Wall ◽  
Endothelial Cell Surface ◽  
Endothelial Surface ◽  
Wide Range ◽  
Storage Organelle ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Changes in the size of cellular organelles are often linked to modifications in their function. Endothelial cells store von Willebrand Factor (vWF), a glycoprotein essential to haemostasis in Weibel-Palade bodies (WPBs), cigar-shaped secretory granules that are generated in a wide range of sizes. We recently showed that forcing changes in the size of WPBs modifies the activity of this cargo. We now find that endothelial cells treated with statins produce shorter WPBs and that the vWF they release at exocytosis displays a reduced capability to recruit platelets to the endothelial cell surface. Investigating other functional consequences of size changes of WPBs, we also report that the endothelial surface-associated vWF formed at exocytosis recruits soluble plasma vWF and that this process is reduced by treatments that shorten WPBs, statins included. These results indicate that the post-exocytic adhesive activity of vWF towards platelets and plasma vWF at the endothelial surface reflects the size of their storage organelle. Our findings therefore show that changes in WPB size, by influencing the adhesive activity of its vWF cargo, may represent a novel mode of regulation of platelet aggregation at the vascular wall.

THE EFFECT OF PHORBOL DIESTERS AND INTERLEUKINS ON THE SECRETION OF VON WILLEBRAND FACTOR BY HUMAN ENDOTHELIAL CELLS IN CULTURE

1987 ◽  
Author(s):  
J C Giddings ◽  
L Shall
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Interleukin 1 ◽  
Interleukin 2 ◽  
Morphological Evidence ◽  
Adhesive Properties ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Human umbilical vein endothelial cells (EC) were cultured in the presence of 4p-phorbol 12-myristate 13-acetate (PMA, 10ug/l), interleukin 1 (IL-1, 1 unit/ml) and interleukin 2 (IL-2, 1 unit/ml), and secretion of von Willebrand factor activity (vWF, Ristocetin co-factor) and von Willebrand factor antigen (vWFAG, ELISA Technique) measured at intervals. Confluent control EC were treated with PMA, IL-1 and IL-2, and the supernatant medium assayed for release of vWF and vWFAg. Treated cells were also examined for vWFAg by immuno-fluorescence. The levels of both vWF and vWFAg in cultures containing IL-1 were significantly higher than those in control cultures after 5-6 days growth. Moreover, vWF and vWFAg increased significantly in the supernatant of confluent control EC incubated further in the presence of IL-1. Furthermore, the characteristic fluorescence pattern of endothelial vWFAg was markedly reduced in EC treated with IL-1. The levels of vWF and vWFAg in cultures containing PMA were also significantly higher than those of control cultures. In these conditions, however, the growth of cells appeared to be enhanced, and confluence was observed after about 6 days in the presence of PMA compared to 9 - 10 days in control cultures. The mean levels of vWF and vWFAg in the supernatant of EC incubated with PMA were higher than the control values but the differences were not statistically significant. Immunofluorescence of PMA-treated cells suggested that vWFAg might be less granular than in control cells but the differences were not as marked as those seen with IL-1. The results of all assays in the presence of IL-2 were not significantly different from those of control cells. In all instances no morphological evidence of endothelial injury was observed and more than 90% of cells remained viable at the termination of cultures. The results indicated that the synthesis and release of vWF were increased in the presence of PMA, and secretion of vWF was stimulated by IL-1. The data suggest that secreted vWF might contribute to the previously reported enhanced procoagulant and adhesive properties of EC treated with these substances.

scholarly journals The roles of protein kinase C and intracellular Ca2+ in the secretion of von Willebrand factor from human vascular endothelial cells

1992 ◽  
Vol 286 (2) ◽  
pp. 631-635 ◽  
Author(s):  
M A Carew ◽  
E M Paleolog ◽  
J D Pearson
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Selective Inhibition ◽  
Von Willebrand ◽  
Willebrand Factor

Secretion of von Willebrand factor (vWf) glycoprotein from storage granules in human umbilical-vein endothelial cells was studied in vitro. Either elevation of intracellular Ca2+ concentration ([Ca2+]i) with a Ca2+ ionophore or activation of protein kinase (PK) C by phorbol 12-myristate 13-acetate caused vWf secretion, and together the agents acted synergistically. However, when vWf release was stimulated by receptor-mediated agonists, selective inhibition of PKC had no effect on histamine-induced secretion and significantly elevated thrombin-induced secretion. Furthermore, ATP, which efficiently elevates [Ca2+]i in these cells, was a very poor effector of vWf release. We conclude that elevation of [Ca2+]i by physiological agonists is necessary for vWf release, but other signalling mechanisms, as yet uncharacterized, but not due to PKC activation, are required for full induction of the secretory pathway.

Von Willebrand Factor in Cultured Human Vascular Endothelial Cells from Adult and Umbilical Cord Arteries and Veins

1986 ◽  
Vol 56 (02) ◽  
pp. 189-192 ◽  
Author(s):  
Pauline B van Wachem ◽  
Jan Hendrik Reinders ◽  
Marijke F van Buul-Wortelboer ◽  
Philip G de Groot ◽  
Willem G van Aken ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Vena Cava ◽  
Ionophore A23187 ◽  
Cultured Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Arteries And Veins

SummaryEndothelial cells were cultured from various human arteries and veins, obtained from adult individuals and from umbilical cords. We compared the storage and secretion of von Willebrand factor by endothelial cells from umbilical veins with that of endothelial cells cultured from a number of adult vessels, including aorta, arteria iliaca, vena saphena magna and vena cava. There were no differences in the way the cultured endothelial cells handled the von Willebrand factor they synthesized. Endothelial cells from the various vessels responded to stimuli in secreting stored von Willebrand factor. The cells also responded to thrombin and ionophore A23187 in producing enhanced amounts of prostacyclin. Thus, cultured umbilical vein endothelial cells have properties that are very similar to those of cultured endothelial cells of various other origins. It is concluded that foetal venous cells provide a representative model for studies of endothelial cell von Willebrand factor biosynthesis and prostacyclin production.

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