Abstract 531: Role of Rna Splicing In Mediating Lineage-specific Expression of the Von Willebrand Factor Gene in the Endothelium

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Lei Yuan ◽  
Lauren Janes ◽  
David Beeler ◽  
Katherine C Spokes ◽  
Joshua Smith ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Rna Splicing ◽  
Coagulation Factor ◽  
Transcriptional Level ◽  
Specific Expression ◽  
First Intron ◽  
Von Willebrand ◽  
Willebrand Factor

We previously demonstrated that the first intron of the human von Willebrand factor (vWF) is required for gene expression in the endothelium of transgenic mice. Based on this finding, we hypothesized that RNA splicing plays a role in mediating vWF expression in the vasculature. To address this question, we employed transient transfection assays in human endothelial cells and megakaryocytes with intron-containing and intronless human vWF promoter-luciferase constructs. Next, we generated knockin mice in which LacZ was targeted to the endogenous mouse vWF locus in the absence or presence of the native first intron or heterologous introns from the human beta-globin, mouse DSCR-1 or hagfish coagulation factor X genes. In both the in vitro assays and the knockin mice, the loss of the first intron of vWF resulted in a significant reduction of reporter gene expression in endothelial cells, but not megakaryocytes. This effect was rescued to varying degrees by the introduction of a heterologous intron. Intron-mediated enhancement of expression was mediated at a post-transcriptional level. Together, these findings implicate a role for intronic splicing in mediating lineage-specific expression of vWF in the endothelium.

scholarly journals Role of RNA splicing in mediating lineage-specific expression of the von Willebrand factor gene in the endothelium

Blood ◽  
2013 ◽  
Vol 121 (21) ◽  
pp. 4404-4412 ◽  
Author(s):  
Lei Yuan ◽  
Lauren Janes ◽  
David Beeler ◽  
Katherine C. Spokes ◽  
Joshua Smith ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Rna Splicing ◽  
Specific Expression ◽  
Factor Gene ◽  
First Intron ◽  
Key Points ◽  
Endothelial Cell Heterogeneity ◽  
Von Willebrand ◽  
Willebrand Factor

Key PointsRNA splicing of the first intron of the von Willebrand factor gene is essential for expression in the endothelium. RNA splicing may play a role in mediating endothelial cell heterogeneity.

Characterization of the Mouse von Willebrand Factor Promoter

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3405-3412 ◽  
Author(s):  
Jiazhen Guan ◽  
Pascale V. Guillot ◽  
William C. Aird
Keyword(s):  
Skeletal Muscle ◽  
Endothelial Cell ◽  
Transgenic Mice ◽  
Von Willebrand Factor ◽  
Dna Sequences ◽  
Specific Expression ◽  
Vascular Bed ◽  
First Intron ◽  
Von Willebrand ◽  
Willebrand Factor

Expression of the von Willebrand factor (vWF) gene is restricted to the endothelial and megakaryocyte lineages. Within the endothelium, expression of vWF varies between different vascular beds. We have previously shown that the human vWF promoter spanning a region between −2182 (relative to the start site of transcription) and the end of the first intron contains information for environmentally responsive, vascular bed-specific expression in the heart, skeletal muscle, and brain. In the present study, we cloned the mouse vWF (mvWF) promoter and studied its function in cultured endothelial cells and transgenic mice. In transient transfection assays, the mvWF gene was found to be regulated by distinct mechanisms in different endothelial cell subtypes. In independent lines of transgenic mice, an mvWF promoter fragment containing DNA sequences between −2645 and the end of the first intron directed endothelial cell-specific expression in the microvascular beds of the heart, brain, and skeletal muscle as well as the endothelial lining of the aorta. In 1 line of mice, reporter gene activity was also detected in bone marrow megakaryocytes. Taken together, these findings suggest that both the mouse and human vWF promoters are regulated by vascular bed-specific mechanisms.

VON WILLEBRAND FACTOR mRNA IS SEVERELY REDUCED IN PIGS WITH HOMOZYGOUS VON WILLEBRAND DISEASE

1987 ◽  
Author(s):  
Q Y Wu ◽  
B R Bahnak ◽  
L Coulombel ◽  
J P Caen ◽  
G Pietu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Limit Of Detection ◽  
Cdna Probe ◽  
Von Willebrand Disease ◽  
Northern Hybridization ◽  
Transcriptional Level ◽  
Total Rna ◽  
Von Willebrand ◽  
Willebrand Factor

Porcine von Willebrand disease (vWD), an autosomal recessive disorder, is similar to some of the severe forms of vWD in humans and is characterized by a prolonged bleeding time and very low or undetectable amounts of von Willebrand factor (vWF) antigen and activity in plasma, platelets and endothelial cells. The molecular events that control the lack of expression of vWF in the vWD pigs is not known and could be at the transcriptional or post-transcriptional level. Lungs from normal and two homozygous vWD pigs were extracted immediately after harvesting of the animals and placed on dry ice. Tissues were homogenized in 6 M guanidinium thiocyanate and RNA isolated by centrifugation through cesium chloride. Total RNA was analyzed by Northern hybridization including dénaturation in glyoxal, electrophoresis in 1.0 % agarose-2.2 M formaldehyde gels and transfer onto nitrocellulose. Messenger RNA was detected with a nick-translated human vWF cDNA probe or a human actin control probe. The vWF probe, cloned from a human lung library, was 2,280 bp in length and spanned nucleotides 960 to 3,240 of the human cDNA. These human probes were considered valid to detect levels of porcine vWF and actin mRNA because they hybridized with restriction enzyme digested genomic DNA from normal and vWD pig leucocytes under conditions of high stringency. The size of the vWF mRNA in the normal pigs after Northern hybridization was approximately 9.0 kb, similar to that of human vWF mRNA, and was easily detectable at the lowest concentration of RNA blotted (5 ug). In contrast, vWF mRNA from vWD pigs was at the lower limit of detection even at 10 ug of total RNA blotted. Nevertheless, although at extremely low levels, vWF mRNA from vWD pigs appeared to be the same size as the normal mRNA. These results agree with observations on the relationship of vWF secreted from 24 hr. cultures of endothelial cells from the pulmonary artery of normal and vWD pigs where the vWF levels were 0.90 and 0.06 U/108 cells, respectively. Therefore, it appears that the very low expression of vWF in the vWD pigs is due to a lack of transcription of the vWF gene. At this time, however, turnover of unstable transcripts in the vWD pigs can not be ruled out.

Co-Trafficking of Coagulation Factor VIII with Von Willebrand Factor Alters the Macromolecular Structure Inside Secretory Weibel-Palade Bodies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 325-325
Author(s):  
Eveline Bouwens ◽  
Marjon Mourik ◽  
Maartje van den Biggelaar ◽  
Jan Voorberg ◽  
Karine Valentijn ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
Coagulation Factor ◽  
Live Cell ◽  
Confocal Imaging ◽  
Macromolecular Structure ◽  
Platelet Binding ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 325 The liver is generally recognized as the major site of coagulation factor (F)VIII synthesis. However, there is now increasing evidence that FVIII can also be synthesized in specific endothelial cells where it is stored with its natural carrier protein von Willebrand factor (VWF) in the Weibel-Palade bodies (WPBs). WPBs have a typical cigar-shaped appearance that most likely originates from the macromolecular organization of VWF multimers into tubules. The tubular storage of VWF is thought to be essential for orderly secretion of VWF strings during activation of endothelial cells. Recently we have shown that expression of FVIII with VWF changes the WPB morphology to spherical vesicles. This finding suggests alterations in the biochemical properties of stored VWF. We now studied in detail the effect of FVIII co-expression on the VWF molecule using a combination of innovative techniques, including correlative light-electron microscopy (CLEM), and live-cell fluorescence microscopy under flow conditions. Analysis of human blood outgrowth endothelial cells (BOECs) expressing human B-domain deleted FVIII-GFP by CLEM revealed that FVIII containing WPBs were electron-dense, spherical structures. These structures contained disorganized short VWF tubules, which was confirmed in 3D by electron tomography. Double immunogold labelling with VWF and GFP antibodies showed that the spherical FVIII containing structures were always positive for VWF. These observations imply that FVIII blocks the expansion of VWF tubules, possibly by binding to the N-terminal VWF domains. As the N-terminal domains are also implicated in the formation of multimers, we therefore investigated whether FVIII affects VWF multimer size. Indeed, multimer analysis showed that VWF secreted by FVIII-GFP transduced BOECs was multimerized to a lesser extent when compared to VWF secreted by non-transduced BOECs. The combined absence of high molecular weight (HMW) VWF multimers and long VWF tubules made us question whether these cells could still release ultra-large VWF (UL-VWF) strings. UL-VWF strings play a key role in bleeding arrest, as platelets adhere to the released VWF string which ultimately leads to the formation of a platelet plug. We examined the release of UL-VWF strings under shear stress from BOECs expressing FVIII-GFP employing live-cell confocal imaging. This technique allowed us to follow FVIII release during exocytosis of WPBs in real-time as well. When we stimulated FVIII-transduced BOECs with histamine, these cells were equally able to release VWF strings as non-transduced BOECs. Although spherical WPBs lacked long VWF tubules and did not secrete HMW multimers, released VWF strings were of similar length as strings secreted by non-transduced BOECs. Surprisingly, released VWF strings were completely covered with FVIII which remained attached to the strings throughout the whole experiment. Another remarkable observation was that platelet binding to the FVIII-covered VWF strings was almost completely absent. We hypothesize that FVIII either shields the A1 domain for platelet binding or causes a conformational change in the VWF strings that prevents platelets from binding to the strings. Our results demonstrate that FVIII co-trafficking with VWF has a major impact on properties of VWF as it reduces the degree of multimerization, shortens tubules and prevents platelets from adhering to strings. This leads us to the conclusion that the macromolecular structure of VWF is considerably altered when FVIII is present in WPBs. Disclosures: No relevant conflicts of interest to declare.

Requirement for Both D Domains of the Propolypeptide in von Willebrand Factor Muitimerization and Storage

1993 ◽  
Vol 70 (06) ◽  
pp. 1053-1057 ◽  
Author(s):  
Agnès M Journet ◽  
Simin Saffaripour ◽  
Denisa D Wagner
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Von Willebrand Factor ◽  
Deletion Mutants ◽  
Relative Importance ◽  
D2 Domain ◽  
Constitutive Secretion ◽  
Von Willebrand ◽  
And Storage ◽  
Willebrand Factor

SummaryBiosynthesis of the adhesive glycoprotein von Willebrand factor (vWf) by endothelial cells results in constitutive secretion of small multimers and storage of the largest multimers in rodshaped granules called Weibel-Palade bodies. This pattern is reproduced by expression of pro-vWf in heterologous cells with a regulated pathway of secretion, that store the recombinant protein in similar elongated granules. In these cells, deletion of the vWf prosequence prevents vWf storage. The prosequence, composed of two homologous domains (D1 and D2), actively participates in vWf multimer formation as well. We expressed deletion mutants lacking either the D1 domain (D2vWf) or the D2 domain (D1vWf) in various cell lines to analyze the relative importance of each domain in vWf muitimerization and storage. Both proteins were secreted efficiently without being retained in the endoplasmic reticulum. Despite this, neither multimerized past the dimer stage and they were not stored. We conclude that several segments of the prosequence are jointly involved in vWf muitimerization and storage.

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.

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