scholarly journals The role of the 5′-flanking region in the cell-specific transcription of the human von Willebrand factor gene

1993 ◽  
Vol 293 (3) ◽  
pp. 641-648 ◽  
Author(s):  
V Ferreira ◽  
Z Assouline ◽  
J L Schwachtgen ◽  
B R Bahnak ◽  
D Meyer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Thymidine Kinase ◽  
Von Willebrand Factor ◽  
Transcriptional Activity ◽  
Cell Types ◽  
Flanking Region ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Cat Gene ◽  
Cpae Cells

Transcriptional regulation of the human von Willebrand factor (vWF) gene was investigated in calf pulmonary artery endothelial (CPAE), HeLa, COS 7 and Hep G2 cells. Various lengths of flanking sequences extending up to 2123 bp 5′ of the transcription initiation site and containing 19 bp of the first exon, were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and these constructs were assayed in transient transfection assays. Sequences up to 89 bp upstream of the cap site showed transcriptional activity in all cell types. Sequences between -147 and -419 bp markedly reduced CAT activity in CPAE cells and abolished it in other cell lines. A domain from -592 to -810 bp generated low levels of expression only in CPAE cells. This transcriptional activity was repressed with constructs containing 1041 to 1240 bp upstream of the cap site. Endothelial cell-specific transcription was restored by a construct that contained 1286 bp upstream of the cap site. The additional 46 bp upstream of the negative regulatory domain were within the 5′ end of an inverse human Alu-family DNA repeat. RNAase-protection assays confirmed the correct transcriptional initiation. The sequence between -89 and -420 contained at least one negative regulatory element able to repress the CAT gene expression controlled by the heterologous thymidine kinase promoter in all cell types. A construct that included the sequence from -89 to -1286 bp increased the transcriptional activity directed by the thymidine kinase promoter only in CPAE cells. These results indicate that negative and positive elements in the 5′-flanking region interact to regulate vWF gene expression.

Processing and Characterization of Recombinant von Willebrand Factor Expressed in Different Cell Types Using a Vaccinia Virus Vector

1992 ◽  
Vol 67 (01) ◽  
pp. 154-160 ◽  
Author(s):  
P Meulien ◽  
M Nishino ◽  
C Mazurier ◽  
K Dott ◽  
G Piétu ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Von Willebrand Factor ◽  
Human Fibroblasts ◽  
Active Form ◽  
Cell Types ◽  
Biologically Active ◽  
L Cells ◽  
Von Willebrand ◽  
Different Cell Types ◽  
Willebrand Factor

SummaryThe cloning of the cDNA encoding von Willebrand factor (vWF) has revealed that it is synthesized as a large precursor (pre-pro-vWF) molecule and it is now clear that the prosequence or vWAgll is responsible for the intracellular multimerization of vWF. We have cloned the complete vWF cDNA and expressed it using a recombinant vaccinia virus as vector. We have characterized the structure and function of the recombinant vWF (rvWF) secreted from five different cell types: baby hamster kidney (BHK), Chinese hamster ovary (CHO), human fibroblasts (143B), mouse fibroblasts (L) and primary embryonic chicken cells. Forty-eight hours after infection, the quantity of vWF antigen found in the cell supernatant varied from 3 to 12 U/dl depending on the cell type. By SDS-agarose gel electrophoresis, the percentage of high molecular weight forms of vWF varied from 39 to 49% relative to normal plasma for BHK, CHO, 143B and chicken cells but was less than 10% for L cells. In all cell types, the two anodic subbands of each multimer were missing. The two cathodic subbands were easily detected only in BHK and L cells. By SDS-PAGE of reduced samples, pro-vWF was present in similar quantity to the fully processed vWF subunit in L cells, present in moderate amounts in BHK and CHO and in very low amounts in 143B and chicken cells. rvWF from all cells bound to collagen and to platelets in the presence of ristocetin, the latter showing a high correlation between binding efficiency and degree of multimerization. rvWF from all cells was also shown to bind to purified FVIII and in this case binding appeared to be independent of the degree of multimerization. We conclude that whereas vWF is naturally synthesized only by endothelial cells and megakaryocytes, it can be expressed in a biologically active form from various other cell types.

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.

Age-Related Increases in Plasma Factor VIII and Von Willebrand Factor in a C57BL/6 Mouse Model Are Associated with Increased Factor VIII and Von Willebrand Factor Gene Expression and Reduced Expression of the Clearance Receptor, Stabilin-2

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4228-4228
Author(s):  
Silvia Albánez ◽  
Alison Michels ◽  
Kate Sponagle ◽  
David Lillicrap
Keyword(s):  
Gene Expression ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Plasma Levels ◽  
Expression Levels ◽  
Age Related ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Old Mice ◽  
Age Related Changes

Abstract Background: Aging is associated with a state of hypercoagulability, as the result of increased concentrations of plasma coagulation proteins. Plasma levels of Factor VIII (FVIII) and von Willebrand factor (VWF) increase with age in humans, but the potential contribution of increases in gene expression with age has not been studied. These two proteins circulate in a non-covalent complex and are cleared together from plasma, hence, a reduction in the expression of their clearance receptors is also a possible pathogenetic explanation. In contrast, plasma levels of ADAMTS13 have been shown to be reduced in later life in humans, but again the mechanism responsible for this age-related pathophysiology is currently unknown. In this study, we utilized a mouse model in which age-related changes in plasma levels of FVIII, VWF and ADAMTS13 were initially documented. Here, we evaluated age-related changes in the gene expression of VWF, FVIII, ADAMTS13 and the clearance receptors low-density lipoprotein receptor-related protein 1 (LRP1), scavenger receptor class A member 5 (SCARA5) and Stabilin-2 (Stab2). Methods: Liver, spleen and lung samples were collected from normal C57BL/6 mice at 9- (n=10), 55- (n=8) and 97-weeks of age (n=15). Also, liver and spleen samples were collected at 3-weeks of age (n=5). Total mRNA was isolated from the tissues and gene expression analysis performed through qRT-PCR by a two-step relative quantification against mouse GAPDH. Expression of murine Factor IX (f9) and Protein C (proc) genes were also measured as positive and negative controls, as the developmental expression of these genes has been extensively studied. The 9-weeks old mice were used as a reference, and expression levels in this group were set as 1. Results were expressed as the fold change median and 95% CI from the 9 week standard group. Data was log10 transformed and compared with a Mann-Whitney test. Additionally, plasma levels of murine VWF, FVIII and ADAMTS13 were measured through ELISA, chromogenic assays and ELISA-based activity assays, respectively, in samples obtained at the same time-points examined for gene expression. Results: Levels of VWF in plasma showed significant increases with age (p<0.0001), reaching a 2-fold increase by 97-weeks. Expression levels increased gradually with age in all three tissues evaluated, reaching a 1.4-fold increase in the lungs (p=0.008), 1.8-fold in the spleen (p=0.01) and 10.3-fold in the liver (p<0.0001) of 97-weeks old mice. When FVIII plasma levels were measured, a similar age-related increase was observed (p<0.0001). Expression levels increased significantly with age in the lungs by 2-fold (1.53-2.68, p=0.002), but no specific age-related changes were observed in liver and spleen. Plasma levels of mouse ADAMTS13 activity showed an opposite pattern to what has been reported for the human protein, with an age-related increase (p<0.0001). When ADAMTS13 gene expression was analyzed in the liver, higher levels were observed in the 3-week old group [1.32 (1.25-1.41), p=0.04], but no significant changes in expression occurred at later time points. Finally, gene expression analysis of LRP1, SCARA5 and Stab2 genes was performed in liver and spleen, the two main organs involved in VWF/FVIII clearance. Expression of these three receptor genes was significantly reduced in both tissues at 3-weeks (<0.04 fold for all estimates). Expression of LRP1 in the liver was an exception to this pattern, with a level that was similar to the 9-week old mice [1.44 (0.96-2.17), p=0.77]. Interestingly, no Stab2 expression was detected in the liver at any point. With aging, no significant changes occurred in SCARA5 and LRP1 gene expression that could be associated with higher plasma levels of VWF/FVIII. However, splenic Stab2 expression significantly decreased with age, reaching a 0.18-fold (0.13-0.25, p=0.02) reduction in the 97-weeks old spleen samples. The positive control gene used (f9) showed no increases in expression with age [1.11 (1.00-1.23), p=0.60], possibly due to strain differences with reported studies, while the negative control gene proc showed no changes [0.87 (0.82-0.93), p=0.28], as expected. Conclusions: Changes in gene expression with increasing age appear to be contributing to the increases in VWF and FVIII plasma levels. Our studies have shown age-related increases in expression of the VWF and FVIII genes and reduced expression of the clearance receptor Stabilin-2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Gene expression profiling and localization of Hoechst-effluxing CD45− and CD45+ cells in the embryonic mouse lung

2005 ◽  
Vol 23 (2) ◽  
pp. 172-181 ◽  
Author(s):  
Simon X. Liang ◽  
Ross Summer ◽  
Xi Sun ◽  
Alan Fine
Keyword(s):  
Gene Expression ◽  
Von Willebrand Factor ◽  
Side Population ◽  
Expression Profiles ◽  
Gene Profiling ◽  
Proteinase 3 ◽  
Side Population Cells ◽  
Embryonic Mouse ◽  
Von Willebrand ◽  
Willebrand Factor

Hoechst-effluxing cells (side population cells) are a rare subset of cells found in adult tissues that are highly enriched for stem and progenitor cell activity. To identify potential stem and progenitor cells during lung development, we generated gene expression profiles for CD45− and CD45+ side population cells in the embryonic day 17.5 lung. We found that side population cells comprise 1% of total embryonic day 17.5 lung cells (55% CD45+, 45% CD45−). Gene profiling data demonstrated an overrepresentation of endothelial genes within the CD45− side population. We used expression of several distinct genes to identify two types of CD45− side population cells: 1) von Willebrand factor+/smooth muscle actin+ cells that reside in the muscular layer of select large vessels and 2) von Willebrand factor+/intercellular adhesion molecule+ cells that reside within the endothelial layer of select small vessels. Gene profiling of the CD45+ side population indicated an overrepresentation of genes associated with myeloid cell differentiation. Consistent with this, culturing CD45+ side population cells was associated with induction of mature dendritic markers (CD86). The microarray results suggested that expression of myeloperoxidase and proteinase-3 might be used to identify CD45+ side population cells. By immunohistochemistry, we found that myeloperoxidase+/proteinase-3+ cells represent a small subset of total CD45+ cells in the embryonic day 17.5 lung and that they reside in the mesenchyme and perivascular regions. This is the first detailed information regarding the phenotype and localization of side population cells in a developing organ.

scholarly journals von Willebrand Factor Permeates Small Vessels in CADASIL and Inhibits Smooth Muscle Gene Expression

2011 ◽  
Vol 3 (1) ◽  
pp. 138-145 ◽  
Author(s):  
Xiaojie Zhang ◽  
He Meng ◽  
Mila Blaivas ◽  
Elisabeth J. Rushing ◽  
Brian E. Moore ◽  
...  
Keyword(s):  
Gene Expression ◽  
Smooth Muscle ◽  
Von Willebrand Factor ◽  
Small Vessels ◽  
Muscle Gene Expression ◽  
Muscle Gene ◽  
Von Willebrand ◽  
Willebrand Factor

Tissue Distribution and Regulation of Murine von Willebrand Factor Gene Expression In Vivo

Blood ◽  
1998 ◽  
Vol 92 (8) ◽  
pp. 2791-2801 ◽  
Author(s):  
Koji Yamamoto ◽  
Vivian de Waard ◽  
Colleen Fearns ◽  
David J. Loskutoff
Keyword(s):  
Gene Expression ◽  
Von Willebrand Factor ◽  
Intraperitoneal Administration ◽  
State Level ◽  
Relative Level ◽  
Small Vessels ◽  
American Society ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWF) is frequently used as a biochemical marker for endothelial cells (ECs). Despite this, little is known about the relative level of expression and regulation of this hemostatic factor in ECs in different vascular beds in vivo. In the present study, we used quantitative reverse transcription polymerase chain reaction and in situ hybridization analysis to study vWF gene expression in murine tissues. Large differences in the level of vWF mRNA were observed when comparing highly vascularized tissues, with the lung and brain containing 5 to 50 times higher concentrations of vWF mRNA than the kidney and liver. In this regard, ECs of small vessels and some microvessels in the lung and brain expressed abundant vWF mRNA, whereas ECs of similar sized vessels in the liver and kidney expressed relatively low levels. In general, significantly higher levels of vWF mRNA and antigen were demonstrated in ECs of larger vessels compared with microvessels and in venous ECs compared with arterial ECs. Although intraperitoneal administration of endotoxin (or tumor necrosis factor-) increased plasma vWF levels, it had variable effects on the steady-state level of vWF mRNA in murine tissues (ie, it decreased vWF mRNA in many tissues, increased it in others, and had little effect on still others). These results indicate that vWF is differentially expressed and regulated in ECs present in different tissues and within the same vascular bed. © 1998 by The American Society of Hematology.

Tissue Distribution and Regulation of Murine von Willebrand Factor Gene Expression In Vivo

Blood ◽  
1998 ◽  
Vol 92 (8) ◽  
pp. 2791-2801 ◽  
Author(s):  
Koji Yamamoto ◽  
Vivian de Waard ◽  
Colleen Fearns ◽  
David J. Loskutoff
Keyword(s):  
Gene Expression ◽  
Von Willebrand Factor ◽  
Intraperitoneal Administration ◽  
State Level ◽  
Relative Level ◽  
Small Vessels ◽  
American Society ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand factor (vWF) is frequently used as a biochemical marker for endothelial cells (ECs). Despite this, little is known about the relative level of expression and regulation of this hemostatic factor in ECs in different vascular beds in vivo. In the present study, we used quantitative reverse transcription polymerase chain reaction and in situ hybridization analysis to study vWF gene expression in murine tissues. Large differences in the level of vWF mRNA were observed when comparing highly vascularized tissues, with the lung and brain containing 5 to 50 times higher concentrations of vWF mRNA than the kidney and liver. In this regard, ECs of small vessels and some microvessels in the lung and brain expressed abundant vWF mRNA, whereas ECs of similar sized vessels in the liver and kidney expressed relatively low levels. In general, significantly higher levels of vWF mRNA and antigen were demonstrated in ECs of larger vessels compared with microvessels and in venous ECs compared with arterial ECs. Although intraperitoneal administration of endotoxin (or tumor necrosis factor-) increased plasma vWF levels, it had variable effects on the steady-state level of vWF mRNA in murine tissues (ie, it decreased vWF mRNA in many tissues, increased it in others, and had little effect on still others). These results indicate that vWF is differentially expressed and regulated in ECs present in different tissues and within the same vascular bed. © 1998 by The American Society of Hematology.

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