Pharmacological and Genetic Inhibition of Autophagy Decreases the Secretion of High Molecular Weight Von Willebrand Factor from Endothelial Cells in Vitro and in Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2770-2770
Author(s):  
Hayley A. Hanby ◽  
X. Long Zheng
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Arterial Thrombosis ◽  
Conflicts Of Interest ◽  
Umbilical Vein ◽  
Vehicle Control ◽  
Autophagic Flux ◽  
Von Willebrand ◽  
Willebrand Factor

The regulation of von Willebrand factor (VWF) processing, packaging, and secretion is important for primary hemostasis. Recently, autophagy has been implicated in modulating VWF maturation and Weibel-Palade body (WPB) morphology. Additionally, treatment of mice and humans with chloroquine (CQ), an anti-malarial agent and pharmacological inhibitor of autophagy, is shown to increase bleeding time. Therefore, we hypothesize that targeting autophagic flux may be therapeutic for arterial thrombotic disorders such as thrombotic thrombocytopenic purpura (TTP). To test this hypothesis, we first treated human umbilical vein endothelial cells (HUVECs) with CQ in culture and showed that CQ dose-dependently decreased VWF antigen levels and multimer sizes in the conditioned medium of histamine-stimulated cells (not shown). Knockdown of an autophagy-related protein (Atg7) with shRNA in HUVECs had similar effect to CQ treatment in VWF secretion and multimer distribution (not shown). More interestingly, daily injections (i.p.) of CQ at 60 mg/kg into Adamts13-/- mice (CAST/Ei strain) for 7 days reduced plasma VWF concentration by more than 60% compared to vehicle control (Fig. 1). Interestingly, VWF secreted from CQ-treated mice remained functional as collagen binding activity/antigen ratio was comparable to vehicle control mice. However, multimer analysis demonstrated the selective lack of ultra large VWF in plasma of Adamts13-/- mice treated with CQ as compared with those treated with vehicle alone (Fig. 2). These results indicate that targeting autophagy pathway with a pharmacological agent, such as CQ, may modulate VWF secretion and, thereby, arterial thrombosis. Our ongoing experiments are to determine the therapeutic efficacy of CQ and other autophagy inhibitors in murine models of arterial thrombosis and TTP. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

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.

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.

scholarly journals EPAC regulates von Willebrand factor secretion from endothelial cells in a PI3K/eNOS-dependent manner during inflammation

2020 ◽  
Author(s):  
Jie Xiao ◽  
Ben Zhang ◽  
Zhengchen Su ◽  
Yakun Liu ◽  
Thomas R. Shelite ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Null Mouse ◽  
Intracellular Camp ◽  
Dependent Manner ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractCoagulopathy is associated with both inflammation and infection, including infection with the novel SARS-CoV-2 (COVID-19). Endothelial cells (ECs) fine tune hemostasis via cAMP-mediated secretion of von Willebrand factor (vWF), which promote the process of clot formation. The exchange protein directly activated by cAMP (EPAC) is a ubiquitously expressed intracellular cAMP receptor that plays a key role in stabilizing ECs and suppressing inflammation. To assess whether EPAC could regulate vWF release during inflammation, we utilized our EPAC1-null mouse model and revealed an increased secretion of vWF in endotoxemic mice in the absence of the EPAC1 gene. Pharmacological inhibition of EPAC1 in vitro mimicked the EPAC1−/− phenotype. EPAC1 regulated TNFα-triggered vWF secretion from human umbilical vein endothelial cells (HUVECs) in a phosphoinositide 3-kinases (PI3K)/endothelial nitric oxide synthase (eNOS)-dependent manner. Furthermore, EPAC1 activation reduced inflammation-triggered vWF release, both in vivo and in vitro. Our data delineate a novel regulatory role of EPAC1 in vWF secretion and shed light on potential development of new strategies to controlling thrombosis during inflammation.Key PointPI3K/eNOS pathway-mediated, inflammation-triggered vWF secretion is the target of the pharmacological manipulation of the cAMP-EPAC system.

Adverse Influence of Cigarette Smoking on the Endothelium

1993 ◽  
Vol 70 (04) ◽  
pp. 707-711 ◽  
Author(s):  
Andrew D Blann ◽  
Charles N McCollum
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Tobacco Smoke ◽  
Lipid Peroxides ◽  
Short Exposure ◽  
Acute Effects ◽  
Adverse Influence ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe effect of smoking on the blood vessel intima was examined by comparing indices of endothelial activity in serum from smokers with that from non-smokers. Serum from smokers contained higher levels of von Willebrand factor (p <0.01), the smoking markers cotinine (p <0.02) and thiocyanate (p <0.01), and was more cytotoxic to endothelial cells in vitro (p <0.02) than serum from non-smokers. The acute effects of smoking two unfiltered medium tar cigarettes was to briefly increase von Willebrand factor (p <0.001) and cytotoxicity of serum to endothelial cells in vitro (p <0.005), but lipid peroxides or thiocyanate were not increased by this short exposure to tobacco smoke. Although there were correlations between von Willebrand factor and smokers consumption of cigarettes (r = 0.28, p <0.02), number of years smoking (r = 0.41, p <0.001) and cotinine (r = 0.45, p <0.01), the tissue culture of endothelial cells with physiological levels of thiocyanate or nicotine suggested that these two smoking markers were not cytotoxic. They are therefore unlikely to be directly responsible for increased von Willebrand factor in the serum of smokers. We suggest that smoking exerts a deleterious influence on the endothelium and that the mechanism is complex.

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 The functions of the A1A2A3 domains in von Willebrand factor include multimerin 1 binding

2016 ◽  
Vol 116 (07) ◽  
pp. 87-95 ◽  
Author(s):  
D'Andra Parker ◽  
Subia Tasneem ◽  
Richard Farndale ◽  
Dominique Bihan ◽  
J. Sadler ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Structural Features ◽  
High Shear ◽  
The Individual ◽  
Static Conditions ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryMultimerin 1 (MMRN1) is a massive, homopolymeric protein that is stored in platelets and endothelial cells for activation-induced release. In vitro, MMRN1 binds to the outer surfaces of activated platelets and endothelial cells, the extracellular matrix (including collagen) and von Willebrand factor (VWF) to support platelet adhesive functions. VWF associates with MMRN1 at high shear, not static conditions, suggesting that shear exposes cryptic sites within VWF that support MMRN1 binding. Modified ELISA and surface plasmon resonance were used to study the structural features of VWF that support MMRN1 binding, and determine the affinities for VWF-MMRN1 binding. High shear microfluidic platelet adhesion assays determined the functional consequences for VWF-MMRN1 binding. VWF binding to MMRN1 was enhanced by shear exposure and ristocetin, and required VWF A1A2A3 region, specifically the A1 and A3 domains. VWF A1A2A3 bound to MMRN1 with a physiologically relevant binding affinity (KD: 2.0 ± 0.4 nM), whereas the individual VWF A1 (KD: 39.3 ± 7.7 nM) and A3 domains (KD: 229 ± 114 nM) bound to MMRN1 with lower affinities. VWF A1A2A3 was also sufficient to support the adhesion of resting platelets to MMRN1 at high shear, by a mechanism dependent on VWF-GPIbD binding. Our study provides new information on the molecular basis of MMRN1 binding to VWF, and its role in supporting platelet adhesion at high shear. We propose that at sites of vessel injury, MMRN1 that is released following activation of platelets and endothelial cells, binds to VWF A1A2A3 region to support platelet adhesion at arterial shear rates.

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.

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