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.

scholarly journals Analysis of von Willebrand factor mRNA from the lung of pigs with severe von Willebrand disease by using a human cDNA probe

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1341-1346 ◽  
Author(s):  
QY Wu ◽  
BR Bahnak ◽  
L Coulombel ◽  
D Kerbiriou-Nabias ◽  
L Drouet ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Cdna Probe ◽  
Von Willebrand Disease ◽  
Northern Analysis ◽  
Cdna Clones ◽  
Total Rna ◽  
Human Cdna ◽  
Actin Mrna ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract To examine the control of porcine von Willebrand factor (vWF) biosynthesis we cloned human vWF complementary DNA (cDNA) and investigated the expression of the vWF gene in lungs from normal pigs and pigs with severe von Willebrand's disease (vWD). Recombinant clones spanning approximately 90% of human vWF cDNA were identified in a lambda gt10 human lung cDNA library by screening with oligonucleotides. One clone spanning nucleotides 960 to 3,240 of human vWF cDNA was used to investigate the steady-state levels of vWF mRNA in lungs from normal pigs and from pigs phenotypically determined to be homozygous for vWD. This clone hybridized with genomic DNA from pig leukocytes when Southern blots were processed under very stringent conditions; therefore, human cDNA clones were considered valid probes to detect porcine mRNA. Northern blot analysis of total RNA from normal pig lung and human umbilical vein endothelial cells identified the vWF mRNA as a molecular species of approximately 9.0 kilobases (kb). A very faint to undetectable band at 9.0 kb in total RNA from lungs of vWD pigs suggested a decreased rate of transcription of the vWF gene. Sucrose density gradient centrifugation of RNA from the vWD pigs confirmed by Northern analysis that the high-molecular weight fractions contained vWF mRNA and at the same size as normal pig mRNA. Dot blot hybridization analysis of vWF and actin mRNA processed under stringent conditions demonstrated that the relative ratio of vWF mRNA to actin mRNA in the vWD pigs varied from 21% to 41% of the ratio observed in normal pigs. Because the amount of vWF mRNA is not correlated to the amount of vWF activity or antigen in plasma of vWD pigs we conclude that posttranscriptional events are also probably involved in abnormal expression of vWF in these animals.

scholarly journals Analysis of von Willebrand factor mRNA from the lung of pigs with severe von Willebrand disease by using a human cDNA probe

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1341-1346
Author(s):  
QY Wu ◽  
BR Bahnak ◽  
L Coulombel ◽  
D Kerbiriou-Nabias ◽  
L Drouet ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Cdna Probe ◽  
Von Willebrand Disease ◽  
Northern Analysis ◽  
Cdna Clones ◽  
Total Rna ◽  
Human Cdna ◽  
Actin Mrna ◽  
Von Willebrand ◽  
Willebrand Factor

To examine the control of porcine von Willebrand factor (vWF) biosynthesis we cloned human vWF complementary DNA (cDNA) and investigated the expression of the vWF gene in lungs from normal pigs and pigs with severe von Willebrand's disease (vWD). Recombinant clones spanning approximately 90% of human vWF cDNA were identified in a lambda gt10 human lung cDNA library by screening with oligonucleotides. One clone spanning nucleotides 960 to 3,240 of human vWF cDNA was used to investigate the steady-state levels of vWF mRNA in lungs from normal pigs and from pigs phenotypically determined to be homozygous for vWD. This clone hybridized with genomic DNA from pig leukocytes when Southern blots were processed under very stringent conditions; therefore, human cDNA clones were considered valid probes to detect porcine mRNA. Northern blot analysis of total RNA from normal pig lung and human umbilical vein endothelial cells identified the vWF mRNA as a molecular species of approximately 9.0 kilobases (kb). A very faint to undetectable band at 9.0 kb in total RNA from lungs of vWD pigs suggested a decreased rate of transcription of the vWF gene. Sucrose density gradient centrifugation of RNA from the vWD pigs confirmed by Northern analysis that the high-molecular weight fractions contained vWF mRNA and at the same size as normal pig mRNA. Dot blot hybridization analysis of vWF and actin mRNA processed under stringent conditions demonstrated that the relative ratio of vWF mRNA to actin mRNA in the vWD pigs varied from 21% to 41% of the ratio observed in normal pigs. Because the amount of vWF mRNA is not correlated to the amount of vWF activity or antigen in plasma of vWD pigs we conclude that posttranscriptional events are also probably involved in abnormal expression of vWF in these animals.

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.

Analysis of the storage and secretion of von Willebrand factor in blood outgrowth endothelial cells derived from patients with von Willebrand disease

Blood ◽  
2013 ◽  
Vol 121 (14) ◽  
pp. 2762-2772 ◽  
Author(s):  
Jiong-Wei Wang ◽  
Eveline A. M. Bouwens ◽  
Maria Carolina Pintao ◽  
Jan Voorberg ◽  
Huma Safdar ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Key Points ◽  
Phenotypic Features ◽  
String Formation ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Multiple Patients

Key Points Isolation of BOECs from multiple patients with VWD is feasible, and the study of BOECs helps explain the pathogenic complexity of VWD. Abnormalities in WPB biogenesis and exocytosis and defects in VWF string formation correlate with the phenotypic features of patients with VWD.

scholarly journals Expression of abnormal von Willebrand factor by endothelial cells from a patient with type IIA von Willebrand disease.

1987 ◽  
Vol 84 (18) ◽  
pp. 6550-6554 ◽  
Author(s):  
R. B. Levene ◽  
F. M. Booyse ◽  
J. Chediak ◽  
T. S. Zimmerman ◽  
D. M. Livingston ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals Molecular studies of von Willebrand disease: reduced von Willebrand factor biosynthesis, storage, and release in endothelial cells derived from patients with type I von Willebrand disease [published erratum appears in Blood 1990 Nov 1;76(9):1901]

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1466-1472 ◽  
Author(s):  
BM Ewenstein ◽  
A Inbal ◽  
JS Pober ◽  
RI Handin
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Messenger Rna ◽  
Umbilical Vein ◽  
Von Willebrand Disease ◽  
Type I ◽  
Molecular Defects ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Endothelial cells were cultured from the umbilical veins of two neonates with type I von Willebrand disease (vWD) and compared with cells cultured in parallel from normal control umbilical veins. In both cases, cultured vWD endothelial cells contained less messenger RNA (mRNA) encoding von Willebrand factor (vWF), and constitutively secreted two- to fourfold less vWF protein than their matched controls. Regulated secretion of stored vWF induced by thrombin or phorbol-12- myristate-13-acetate (PMA) was also diminished in vWD cells. Both the mRNA and protein produced by each of these type I vWD cells appeared to be of normal size. However, despite the diminished size of the vWF storage pool, electron microscopy of endothelial cells in situ showed normal appearing vWF storage organelles (Weibel-Palade bodies). These studies show that cultured umbilical vein endothelial cells can be used to explore the molecular defects in type I and perhaps other forms of vWD, and suggest that at least some forms of type I vWD are caused by diminished mRNA transcription or subsequent translation due to a defective vWF allele.

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