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.

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 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.

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.

Deficiency of the VWF-Cleaving Protease ADAMTS13 Results in Increased Leukocyte Rolling and Adhesion in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 290-290 ◽  
Author(s):  
Anil K. Chauhan ◽  
Janka Kiucka ◽  
Alexander Brill ◽  
Meghan T. Walsh ◽  
Denisa D. Wagner
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Leukocyte Adhesion ◽  
Surface Expression ◽  
Leukocyte Rolling ◽  
Type I ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adherent Leukocytes

Abstract von Willebrand factor (VWF) is synthesized in megakaryocytes and endothelial cells and stored in a-granules and Weibel-Palade bodies, respectively. VWF levels are elevated in both chronic and acute inflammation. ADAMTS13 (A D isintegrin-like A nd M etalloprotease with T hrombo s pondin type I repeats-13) is a metalloprotease that cleaves ultra large von Willebrand factor (ULVWF) multimers quickly after its release from endothelium. Recent studies have found that VWF promotes leukocyte adhesion in vitro and that ADAMTS13 activity is reduced in inflammation and sepsis. We hypothesized that by cleaving ULVWF multimers, ADAMTS13 not only inhibits thrombosis, but also attenuates leukocyte rolling and adhesion. Using intravital microscopy, we found more leukocyte rolling/min on the unstimulated veins in Adamts13-/- mice (Mean ± SE: 98 ± 16) compared to WT (Mean ± SE: 35 ± 6, P<0.001), n=18–20 from 10–11 mice per group. This process was dependent on VWF because the number of leukocytes rolling in Adamts13-/-/Vwf-/- veins was similar to that in Vwf-/-. Significantly increased soluble P-selectin and VWF concentrations were found in the plasma of Adamts13-/- compared to WT mice as quantitated by ELISA. In addition, endothelial P-selectin surface expression was increased in Adamts13-/- mice compared to WT. These results suggest elevated release of Weibel-Palade bodies in Adamts13-/- mice. Notably, circulating platelets were not activated in the absence of ADAMTS13. Upon stimulation of the mesentery with histamine, leukocyte rolling was slower in Adamts13-/- veins compared to WT. Furthermore, upon stimulation with the inflammatory cytokine TNF-alpha (i.v) 3.5 h prior to surgery, the number of leukocytes adhering/250 um was significantly increased in microvenules (diameter of 25–30 um) of Adamts13-/- mice (Mean ± SD: 21 ± 6) compared to WT (Mean ± SD: 12 ± 5, P<0.001), n=10–11 mice per group. This firm adhesion was also dependent on VWF because the number of adherent leukocytes in veins of Adamts13-/-/Vwf-/- was similar to Vwf-/-. Our studies indicate a crucial role for ADAMTS13 in preventing excessive spontaneous Weibel-Palade secretion and in attenuating leukocyte rolling and adhesion to ultra large VWF presented by endothelial cells during inflammation.

scholarly journals Perturbation of human endothelial cells by thrombin or PMA changes the reactivity of their extracellular matrix towards platelets.

1987 ◽  
Vol 104 (3) ◽  
pp. 697-704 ◽  
Author(s):  
P G de Groot ◽  
J H Reinders ◽  
J J Sixma
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
De Novo ◽  
The Matrix ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets ◽  
De Novo Protein Synthesis

In this study we have examined the influence of perturbation of endothelial cells on the amounts of fibronectin and von Willebrand factor in their extracellular matrix and the consequences of a changed composition of the matrix on platelet adhesion. For this purpose, we have used an in vitro perfusion system with which we can investigate the interactions of platelets in flowing blood with cultured endothelial cells and their extracellular matrix (Sakariassen, K. S., P. A. M. M. Aarts, P. G. de Groot, W. P. M. Houdgk, and J. J. Sixma, 1983, J. Lab. Clin Med. 102:522-535). Treatment of endothelial cells with 0.1-1.0 U/ml thrombin for 2 h increased the reactivity of the extracellular matrix, isolated after the thrombin treatment, towards platelets by approximately 50%. The increased reactivity did not depend on de novo protein synthesis but was inhibited by 3-deazaadenosine, an inhibitor of phospholipid methylation, which also inhibits the stimulus-induced instantaneous release of von Willebrand factor from endothelial cells. However, no changes in the amounts of von Willebrand factor and fibronectin in the matrix were detected. Thrombin may change the organization of the matrix proteins, not the composition. When endothelial cells were perturbed with the phorbol ester PMA or thrombin for 3 d, the adhesion of platelets to the extracellular matrix of treated cells was strongly impaired. This impairment coincided with a decrease in the amounts of von Willebrand factor and fibronectin present in the matrix. These results indicate that, after perturbation, endothelial cells regulate the composition of their matrix, and that this regulation has consequences for the adhesion of platelets.

Reversible Self-Assembly of Weibel-Palade Body-Like Tubules from Recombinant N-Terminal Domains of von Willebrand Factor.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 291-291 ◽  
Author(s):  
Ren-Huai Huang ◽  
Ying Wang ◽  
Robyn Roth ◽  
Xiong Yu ◽  
Angie R. Purvis ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Building Blocks ◽  
Reconstruction Method ◽  
Thin Sections ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Weibel-Palade bodies (WPBs) are elongated secretory granules of endothelial cells that are packed with tubules composed of von Willebrand factor (VWF), a multimeric protein required for hemostasis. Disruption of tubular packing prevents the orderly secretion of VWF multimers and blocks the subsequent binding of platelets. The cigar-like shape and tubular cross section of WPBs are conserved in all vertebrates, but little is known about how VWF specifies this packing arrangement. Starting from recombinant 82 kDa VWF propeptide (domains D1D2) and 114 kDa disulfide-bonded D’D3 dimer, we now have assembled tubules reversibly in vitro with the same dimensions as VWF tubules in WPBs. Assembly was induced at pH 6.2, reversed at pH 7.4, and required Ca2+. Recombinant D’D3 dimers did not self-associate at pH 7.4 or pH 6.2, with or without Ca2+. Without Ca2+, VWF propeptide did not bind to D’D3 dimers. At pH 7.4, with Ca2+, VWF propeptide formed noncovalent 160 kDa dimers and, when mixed with D’D3 dimers, assembled a 280 kDa complex of two propeptides and one D’D3 dimer as shown by gel filtration chromatography and multi-angle light scattering. Lowering the pH to 6.2 caused the formation of >3 MDa aggregates with the same stoichiometry, which dissociated upon adding EDTA or raising the pH to 7.4. Quick-freeze deep-etch EM showed that the large aggregates are hollow right-handed tubular helices. The iterative helical real space reconstruction method was used to make 3D reconstructions of the tubules at 22 Å resolution from negative stain EM images (Figure, left). Tubules consist of a right-handed helix with axial rise of 26.2 Å and twist of 85.6 degrees per subunit, or 4.2 subunits per 11 nm turn. The dimensions (outside diameter 25 nm, inside diameter 12 nm) are similar to those of tubules in WPBs in thin sections of endothelial cells by transmission EM (Figure, right and its insert). Each subunit contains one D’D3 dimer flanked by two D1D2 propeptides (Figure, center). Each D’D3 dimer makes a total of six contacts with D1D2 domains. Each D1D2 propeptide makes three contacts with D’D3 and just one end-to-end homotypic contact. The spatial arrangement of these building blocks and inter-domain contacts in tubules suggest a model by which decreasing pH along the secretory pathway coordinates the formation of intersubunit disulfide bonds with the tubular packaging of VWF multimers. Within the WPB, Ca2+-dependent and pH-dependent binding of D1D2 to D’D3 domains stabilizes the packing of VWF multimers into tubules, which behave as constrained springs. Upon secretion, the increased pH weakens these constraints and permits the helical tubules to unfurl into flowing blood without tangling. Figure Figure

Von Willebrand Factor (VWF) Regulates Angiogenesis: Studies in Blood-Derived Endothelial Progenitor Cells From Von Willebrand's Disease Patients and VWF-Deficient Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5311-5311
Author(s):  
Richard D Starke ◽  
Koralia Paschalaki ◽  
Francesco Ferraro ◽  
Thomas A J McKinnon ◽  
Nicola H Dryden ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Vascular Network ◽  
Vascular Density ◽  
Type I ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
In Vitro Angiogenesis ◽  
Deficient Mice

Abstract Abstract 5311 Dysregulation of angiogenesis is implicated in many diseases. Von Willebrand factor (VWF), a large plasma glycoprotein essential for normal haemostasis is synthesized by endothelial cells (EC) and megakaryocytes. Raised VWF plasma levels are a risk factor for arterial thrombosis, whilst deficiency of VWF causes Von Willebrand disease (VWD), the most common congenital bleeding disorder in man. VWD can be associated with angiodysplasia, vascular malformations linked to defective angiogenesis which are responsible for intractable bleeding. We recently showed that VWF is involved in angiogenesis. Inhibition of VWF expression in human umbilical vein EC (HUVEC) with specific siRNA resulted in increased in vitro angiogenesis on Matrigel, proliferation and migration. Mechanism studies implicated the endothelial VWF receptor, integrin αvβ3 and the angiogenesis regulator angiopoietin-2. The findings were confirmed in EC from VWD patients and in VWF-deficient mice. Blood outgrowth endothelial cells (BOEC) isolated from peripheral blood of patients with VWD showed decreased VWF release, compared to control BOEC, and increased in vitro angiogenesis, migration and proliferation, similar to what observed with VWF siRNA-treated HUVEC. In vivo studies using the matrigel model and imaging of blood vessels in the ear showed increased angiogenesis and vascular network in VWF-deficient mice compared to controls. Recent studies in patients BOEC and in VWF deficient mice provide new insight into the complexity of this phenotype. We have used the mouse model of post-natal angiogenesis in the retina to carry out detailed analysis of angiogenic networks in the VWF-deficient mouse, and found increased vascular density and defective vascular network. Moreover, using BOEC from patients with type I and type II VWD, we have studied VWF intracellular distribution by immunofluorescence confocal analysis and found patterns of expression that point to a variety of defects in synthesis, storage and secretion. These studies define a new function for VWF, which may have clinical implications for VWD and for patients at risk of CV disease. Moreover, studies with BOEC from VWF patients provide a novel understanding of the physiopathology of this disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Rickettsia rickettsii infection of cultured endothelial cells induces release of large von Willebrand factor multimers from Weibel-Palade bodies

Blood ◽  
1991 ◽  
Vol 78 (10) ◽  
pp. 2595-2602 ◽  
Author(s):  
LA Sporn ◽  
RJ Shi ◽  
SO Lawrence ◽  
DJ Silverman ◽  
VJ Marder
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Culture Medium ◽  
Spotted Fever ◽  
Clinical Manifestations ◽  
Tissue Ischemia ◽  
Rickettsia Rickettsii ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The clinical manifestations of Rocky Mountain spotted fever (RMSF) result from Rickettsia rickettsii (R rickettsii) infection of endothelial cells and are mediated by pathologic changes localized to the vessel, including in situ thrombosis and tissue ischemia. This study uses in vitro infection of cultured human umbilical vein endothelial cells with R rickettsii to test the hypothesis that such infection induces von Willebrand factor (vWF) release from Weibel- Palade bodies, a process that could contribute to thrombotic changes. At 24 hours postinfection, there was an increase in metabolically prelabeled large multimers of vWF in the culture medium, with a concomitant decrease of these forms in the cell lysate samples. This release reaction was specific for the large multimer pool of vWF, localized to Weibel-Palade bodies, because no change in the distribution of dimeric forms between cells and culture medium was detected. Double-label immunofluorescence staining showed an inverse correlation between the number of R rickettsii and the number of Weibel- Palade bodies in infected cells. Cell lysis was minimal at 24 hours postinfection, as no detectable intracellular precursor forms (molecular weight 260,000) of vWF were released into the culture medium, there was no decrease in cell viability as measured by trypan blue exclusion, and no increase in 51Cr-release into the culture medium was observed when compared with uninfected controls. Release was likely a direct effect of the intracellular presence of the organism, rather than due to a noxious soluble factor such as endotoxin, because culture medium conditioned by infected endothelial cells was ineffective at inducing release in uninfected endothelial cell cultures. In summary, in vitro infection of endothelial cells by R rickettsii induces release of Weibel-Palade body contents, a process that may contribute to the pathogenesis of RMSF.

DIVERGENT PATES OF VON WILLEBRAND FACTOR AND ITS PROPOLYPEPTIDE (VON WILLEBRAND ANTIGEN II) AFTER SECRETION FROM ENDOTHELIAL CELLS

1987 ◽  
Author(s):  
D D Wagner ◽  
P J Fay ◽  
L A Sporn ◽  
S Sinha ◽  
S O Lawrence ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Culture Medium ◽  
Platelet Adhesion ◽  
Human Foreskin Fibroblasts ◽  
Covalently Linked ◽  
Von Willebrand ◽  
Willebrand Factor

The intracellular site of cleavage of pro-von Willebrand factor subunit and the subsequent fate of the propolypeptide (von Willebrand antigen II) and of the mature von Willebrand factor (vWf) were investigated. Both the propolypeptide, which was found to be a homodimer of non-covalently linked subunits, and mature vWf were released from Weibel-Palade bodies of endothelial cells following stimulation with secretagogues. The stoichiometry of the two proteins in the releasate was essentially equimolar. This indicates that vWf and the propolypeptide were packaged into the Weibel-Palade bodies as one unit, pro-vWf, and that the proteolytic cleavage of pro-vWf is likely to be a post-Golgi event. The association of prosequences into dimers provides support for their hypothetical role in the multimerization process. After secretion, the two proteins were distributed differently, as based on the following observations. The propolypeptide did not associate with vWf in the culture medium, did not co-distribute with vWf in the extracellular "patches of release" on stimulated endothelial cells, and was not detected in the endothelial cell extracellular matrix, which did contain vWf. Additionally, in contrast to vWf, the propolypeptide did not bind to matrix of human foreskin fibroblasts. Since the propolypeptide does not associate with vWf and does not interact witji extracellular matrices in vitro, it is highly unlikely that it would promote platelet adhesion to subendothelium in vivo.

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