Von Willebrand Factor Promoter Research Articles

Retracted: Correlation Analysis of DNA Methylation in the von Willebrand Factor Promoter Region and the Risk of Unexplained Recurrent Hemophilia: Systematic Review and Meta-Analysis.

[This retracts the article DOI: 10.1155/2022/3977289.].

Correlation Analysis of DNA Methylation in the von Willebrand Factor Promoter Region and the Risk of Unexplained Recurrent Hemophilia: Systematic Review and Meta-Analysis.

This study systematically reviewed the effect of DNA methylation in the promoter region of the coagulation factor vWF gene on the risk of unexplained recurrent hemophilia. PubMed, Medline, Web of Science, and other computers were used to search the database, and the statistical randomized controlled trials of coagulation factor vWF in the risk analysis of unknown recurrent hemophilia were collected. The Cochrane systematic evaluation method was used to evaluate the quality of the included kinds of literature, and Revman5 software was used to sort out and analyze the kinds of literature. Meta-analysis showed that there was a statistical difference between the experimental group and the control group in case fatality rate (OR = 1.76, 95% CI (1.29, 2.39), P=0.0003, I2 = 0%, Z = 3.58), adverse events (OR = 2.38, 95% CI (1.65, 3.45), P < 0.00001, I2 = 0%, Z = 4.60), incidence of joint hemorrhage (OR = 2.52, 95% CI (1.62, 3.91), P < 0.00001, I2 = 0%, Z = 4.12), incidence of subcutaneous stasis (OR = 1.76, 95% CI (1.26, 2.45), P=0.0009, I2 = 5%, Z = 3.33), and hematoma volume (OR = 1.78, 95% CI (1.32, 2.40), P=0.0001, I2 = 23%, Z = 3.80). DNA methylation in the promoter region of the coagulation factor vWF gene was significantly associated with the risk of unexplained recurrent hemophilia. Whether demethylation can improve the bleeding index of patients with recurrent hemophilia remains to be further explored.

Endothelial cells of different organs exhibit heterogeneity in von Willebrand factor expression in response to hypoxia

Background and aimsWe have previously demonstrated that in response to hypoxia, von Willebrand factor (VWF) expression is upregulated in lung and heart endothelial cells both in vitro and in vivo, but not in kidney endothelial cells. The aim of our current study was to determine whether endothelial cells of different organs employ distinct molecular mechanisms to mediate VWF response to hypoxia. MethodsWe used cultured human primary lung, heart and kidney endothelial cells to determine the activation of endogenous VWF as well as exogenously expressed VWF promoter in response to hypoxia. Chromatin immunoprecipitation and siRNA knockdown analyses were used to determine the roles of VWF promoter associated transacting factors in mediating its hypoxia response. Platelet aggregates formations in vascular beds of mice were used as a marker for potential functional consequences of hypoxia-induced VWF upregulation in vivo. ResultsOur analyses demonstrated that while Yin Yang 1 (YY1) and specificity protein 1 (Sp1) participate in the hypoxia-induced upregulation of VWF specifically in lung endothelial cells, GATA6 mediates this process specifically in heart endothelial cells. In both cell types, the response to hypoxia involves the decreased association of the NFIB repressor with the VWF promoter, and the increased acetylation of the promoter-associated histone H4. In mice exposed to hypoxia, the upregulation of VWF expression was concomitant with the presence of thrombi in heart and lung, but not kidney vascular beds. ConclusionsHeart and lung endothelial cells demonstrated VWF upregulation in response to hypoxia, using distinct mechanisms, while this response was lacking in kidney endothelial cells.

Epigenetic Profiles of Primary Endothelial Cells from Patients with Low VWF Levels

BackgroundVon Willebrand disease (VWD) type 1 is characterized by low von Willebrand factor (VWF) levels and mucocutaneous bleeding (MCB). Approximately 50% of patients with VWD type 1 exhibit mutations in VWF. However, a large number of patients with VWF levels between 30-50 IU/dL do not show mutations in VWF indicating that other mechanisms are involved. Blood outgrowth endothelial cells (BOECs) are a source of donor-specific endothelial cells and have demonstrated impairments in VWF release and packaging in patients with VWD. BOECs have not been evaluated in individuals with low VWF levels.Hypothesis/ObjectiveWe hypothesize that BOECs from individuals with low VWF levels will reveal unique VWF and genome wide epigenetic signatures that may explain the altered plasma VWF levels seen in these patients.MethodsBOEC Derivation: Patients with low VWF levels and MCB (30-50 IU/dL) were enrolled in an IRB-approved study. The mononuclear layer from whole blood was isolated and plated onto collagen coated plates. After extended incubation, the presence of BOECs was confirmed by visual morphology and flow cytometry.VWF Transcriptional Analysis: 9 cells lines including: a) 2 BOEC cell lines from control individuals and a HUVEC cell line and c) BOECs from individuals with low VWF, were assayed via single cell RNA sequencing. Bioinformatic analysis included generalized transcriptional expression and single cell expression of VWF. RNA-sequencing expression data was filtered according to the following standardized algorithm. Cells that were defined as monocytes (TYROBP expression > 2 copies) were excluded. Following monocyte exclusions, cells were determined to be of endothelial origin if they demonstrated the presence of PECAM1, CDH5, ROBO4, ESAM, TIE1, or NOTCH4 transcripts, as previously reported by Butler et al. (Cell Reports, 2016).Epigenetic Profiling:Genomic DNA was extracted from BOECs and from peripheral leukocytes (paired to the BOEC draw sample) and analyzed for DNA methylation via an Illumina 850K methylation array.ResultsBOEC Derivation:A total of eight BOEC lines were generated, 6 from individuals with MCB and VWF levels between 30-50 IU/dL (5:1 female: male ratio, age range 11-54 years) and 2 from healthy controls (2 female, age range 22-39 years) with normal VWF levels and no symptoms of MCB.VWF Expression is decreased in Low VWF Samples: Overall transcript expression of VWF was significantly decreased in low VWF BOEC samples (5.341 transcripts/cell) vs. control endothelial cells (9.076 transcripts/cell), P <0.0001.Generalized Methylation Profiling:Via adjusted P-values, there were 129 methylation sites across multiple genes that were differentially methylated in Low VWF BOECs vs. control endothelial cells. A cluster plot demonstrates that the two control BOEC samples were generally clustered as compared to the other samples (Figure 1A).VWF Specific Methylation: The Illumina 850K array covers 70 prospective methylation sites in VWF, ranging from upstream of the transcriptional start site through the length of the gene. A previous report demonstrated that differences in 8 methylation sites in the VWF promoter correlated with VWF expression (Yuan et al. Nature Communications 2016). 7 of these sites are covered in our assay. Across all of those 7 sites, there was significant increased methylation of the CpG islands in the Low VWF BOECs when compared to the control endothelial cells (Figure 1B).Stability of VWF Methylation:To ensure that the isolation and culture of BOECS does not significantly affect the methylation status of VWF, we conducted a Pearson correlation analysis and demonstrated that peripheral leukocyte (at time of blood draw) and BOEC methylation is highly correlated at VWF specific methylation sites (R2 0.6, P = 0.0004) (Figure 1C).ConclusionsSingle cell RNA sequencing and genome wide methylation assays of BOECs from individuals with low VWF reveal significant differences in generalized methylation status when compared to BOECs from individuals with normal VWF levels and HUVECs. There is transcriptional downregulation of VWF in low VWF BOECs that is associated with hypermethylation of 7 specific VWF CpG sites in the VWF promoter. Additional sites are being evaluated. Finally, we validated the methylation status of BOECs by demonstrating high correlation with the methylation status of leukocytes from the same individuals. DisclosuresNg:Shire: Consultancy; CSL Behring: Consultancy.

A role of stochastic phenotype switching in generating mosaic endothelial cell heterogeneity.

Previous studies have shown that biological noise may drive dynamic phenotypic mosaicism in isogenic unicellular organisms. However, there is no evidence for a similar mechanism operating in metazoans. Here we show that the endothelial-restricted gene, von Willebrand factor (VWF), is expressed in a mosaic pattern in the capillaries of many vascular beds and in the aorta. In capillaries, the mosaicism is dynamically regulated, with VWF switching between ON and OFF states during the lifetime of the animal. Clonal analysis of cultured endothelial cells reveals that dynamic mosaic heterogeneity is controlled by a low-barrier, noise-sensitive bistable switch that involves random transitions in the DNA methylation status of the VWF promoter. Finally, the hearts of VWF-null mice demonstrate an abnormal endothelial phenotype as well as cardiac dysfunction. Together, these findings suggest a novel stochastic phenotype switching strategy for adaptive homoeostasis in the adult vasculature.

Von Willebrand factor is reversibly decreased during torpor in 13-lined ground squirrels

During torpor in a hibernating mammal, decreased blood flow increases the risk of blood clots such as deep vein thrombi (DVT). In other animal models platelets, neutrophils, monocytes and von Willebrand factor (VWF) have been found in DVT. Previous research has shown that hibernating mammals decrease their levels of platelets and clotting factors VIII (FVIII) and IX (FIX), increasing both bleeding time and activated partial thromboplastin time. In this study, FVIII, FIX and VWF activities and mRNA levels were measured in torpid and non-hibernating ground squirrels (Ictidomys tridecemlineatus). Here, we show that VWF high molecular weight multimers, collagen-binding activity, lung mRNA and promoter activity decrease during torpor. The VWF multimers reappear in plasma within 2 h of arousal in the spring. Similarly, FIX activity and liver mRNA both dropped threefold during torpor. In contrast, FVIII liver mRNA levels increased twofold while its activity dropped threefold, consistent with a post-transcriptional decrease in FVIII stability in the plasma due to decreased VWF levels. Finally, both neutrophils and monocytes are decreased eightfold during torpor which could slow the formation of DVT. In addition to providing insight in how blood clotting can be regulated to allow mammals to survive in extreme environments, hibernating ground squirrels provide an interesting model for studying.

Epigenetic Modification of Von Willebrand Factor (VWF) Leads to its Expression in Cancer Cells with Increased Metastatic Activity

VWF is an adhesive procoagulant protein that is exclusively expressed in endothelial cell (EC) and megakaryocytes. It is key initiator of blood clotting cascade. Increased plasma levels of VWF and alterations in coagulation system in patients with metastasis and cancer progression are reported. We hypothesised that a subpopulation of some cancer cells of non‐endothelial origin may acquire VWF expression and as a result develop enhanced metastatic potential.RT‐PCR, western blot and Immunoflourescent (IF) analyses showed significant levels of VWF expression in an osteosarcoma (Saos2) and glioma (U251, M016, M049) cell lines. Chromatin IP assays demonstrated a similar pattern of transcription factors binding as is observed for VWF gene in EC. The epigenetic modification analysis showed that histone modification of the VWF promoter in glioma (U251, M016, M049) and Saos2 is consistent with transcriptionally active VWF promoter. In vitro cell‐cell interaction analyses showed that cancer cell lines expressing VWF, exhibited increased affinity to adhere to the platelets and an EC monolayer under sheer stress. IF analyses of human glioma tumor samples, demonstrated VWF expression in some cells of non‐endothelial origin in the tumor region, highly suggestive of VWF expression in a subset of glioma cancer cells. Chick Chorioallantoic Membrane assay showed increased extravasation and metastatic lesions for cancer cells expressing VWF.A subpopulation of cancer cells of non‐endothelial origin acquires denovo expression of VWF as a result of epigenetic modification of the VWF promoter. Acquired VWF expressions by cancer cells increase their adhesive properties and may contribute to their metastatic potential.

GENERATION AND UTILIZATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS-DERIVED-IPS FOR STUDYING ENDOTHELIAL GENE REGULATION

Damage to vascular endothelial cells (EC) is the core of various vascular diseases such as stroke and cardiovascular complications. Understanding molecular mechanisms that govern endothelial gene expression and function is crucial for developing novel therapeutic approaches for vascular endothelial dysfunction-related complications. Induced pluripotent stem cells (iPS) provide a valuable in vitro system to study the molecular processes, including EC-specific gene regulation, that contribute to establishment of EC phenotype. We aimed to study endothelial gene regulation by generating iPS from human umbilical vein EC (HUVECs) and differentiating the resulting iPS back into EC. This model provides a system with a homogenous genetic Background to explore how EC phenotype is revoked (HUVEC to iPS), and reestablished (iPS to EC). Colonies (iPS) were generated using originally reported transcription factors. Quantitative RT-PCR and immunofluorescence analysis were used to characterize iPS and cell lineages derived from iPS. Pluripotency of the iPS were demonstrated by generation of embryoid bodies (EB) and detection of the three germ layer markers. Additionally a novel method was used to directly differentiate iPS into neuronal cell lineages with high efficiency without the use of EB. Next EB generated from iPS were induced to differentiate into EC, thus establishing the EC to iPS back to EC system for analyses. In this system we explored the pattern of activation and repression of a highly EC-specific gene, von Willebrand factor (VWF), as well as transacting factors and cofactors that regulate the VWF promoter activity. Our analyses demonstrated that expression of transacting factors that function as activators of the VWF promoter mirrored that of VWF gene, while those functioning as repressors where either reversed or not significantly altered. Temporal expression of activators were observed during various stages of differentiation. Specifically during the progression from EB to EC we observed upregulation of GATA6 followed by Ets transactivators. Furthermore, the ability of iPS to incorporate into microvasculature was demonstrated in a mouse model of kidney vascular injury. Generation of iPS from EC and differentiation of iPS back to EC provides a unique opportunity to explore the molecular mechanisms that are involved in establishment of EC specific gene regulation and consequently the establishment of EC phenotype. This system will also provide an opportunity for exploring the potential of these EC derived iPS and their differentiated EC towards developing cell- therapy approaches for vascular diseases.

Performance Related Factors Are the Main Determinants of the von Willebrand Factor Response to Exhaustive Physical Exercise

BackgroundPhysical stress triggers the endothelium to release von Willebrand Factor (VWF) from the Weibel Palade bodies. Since VWF is a risk factor for arterial thrombosis, it is of great interest to discover determinants of VWF response to physical stress. We aimed to determine the main mediators of the VWF increase by exhaustive physical exercise.Methods105 healthy individuals (18–35 years) were included in this study. Each participant performed an incremental exhaustive exercise test on a cycle ergometer. Respiratory gas exchange measurements were obtained while cardiac function was continuously monitored. Blood was collected at baseline and directly after exhaustion. VWF antigen (VWF:Ag) levels, VWF collagen binding (VWF:CB) levels, ADAMTS13 activity and common variations in Syntaxin Binding Protein-5 (STXBP5, rs1039084 and rs9399599), Syntaxin-2 (STX2, rs7978987) and VWF (promoter, rs7965413) were determined.ResultsThe median VWF:Ag level at baseline was 0.94 IU/mL [IQR 0.8–1.1] and increased with 47% [IQR 25–73] after exhaustive exercise to a median maximum VWF:Ag of 1.38 IU/mL [IQR 1.1–1.8] (p<0.0001). VWF:CB levels and ADAMTS13 activity both also increased after exhaustive exercise (median increase 43% and 12%, both p<0.0001). The strongest determinants of the VWF:Ag level increase are performance related (p<0.0001). We observed a gender difference in VWF:Ag response to exercise (females 1.2 IU/mL; males 1.7 IU/mL, p = 0.001), which was associated by a difference in performance. Genetic variations in STXBP5, STX2 and the VWF promoter were not associated with VWF:Ag levels at baseline nor with the VWF:Ag increase.ConclusionsVWF:Ag levels strongly increase upon exhaustive exercise and this increase is strongly determined by physical fitness level and the intensity of the exercise, while there is no clear effect of genetic variation in STXBP5, STX2 and the VWF promoter.

Hypoxia Results in Upregulation and De Novo Activation of Von Willebrand Factor Expression in Lung Endothelial Cells

Increased von Willebrand factor (VWF) levels in lungs are associated with diseases such as pulmonary hypertension. The objective of our study was to determine the mechanism of increased VWF levels in conditions, such as hypoxia, which contribute to pulmonary hypertension. We have previously reported generation of transgenic mice that express LacZ transgene under the regulation of lung- and brain-specific transcriptional regulatory elements of the VWF gene. Hypoxia exposure of these transgenic mice resulted in increased VWF and LacZ mRNA levels as well as redistribution of their expression from primarily larger vessels in the lungs to microvessels. Exposure of cultured lung microvascular endothelial cells to hypoxia demonstrated that VWF upregulation was accompanied by increased platelet binding. Transcription upregulation was mediated through inhibition of the repressor nuclear factor-IB association with the VWF promoter, and increased nuclear translocation of the transcription factor YY1 and association with its cognate binding site on the VWF gene. Knockdown of YY1 expression abolished the hypoxia-induced upregulation and reduced basal level of VWF. These analyses demonstrate that hypoxia induces a phenotypic shift, accompanied by modulation of nuclear factor-IB and YY1 activities, in microvascular endothelial cells of the lungs to support VWF promoter activation.

002 Mechanism of Hypoxia Induction of VWF

002 Mechanism of Hypoxia Induction of VWF

470 Hypoxia-induced pulmonary hypertension in mice modifies the expression pattern of von willebrand factor gene in lung endothelial cells

470 Hypoxia-induced pulmonary hypertension in mice modifies the expression pattern of von willebrand factor gene in lung endothelial cells

New Insight into the Hypercoagulability of Cushing’s Syndrome

Background: Hypercoagulability and a tendency for thromboembolic complications are reported in Cushing’s syndrome (CS). The hypercoagulability is due mainly to the cortisol-induced increase in von Willebrand factor (VWF) and factor VIII. This is not a constant feature of CS, however; it depends on particular single nucleotide polymorphism (SNP) haplotypes in the VWF gene promoter: haplotype 1 (–3268G/–2709C/–2661A/–2527G) confers a greater risk of VWF upregulation by cortisol than haplotype 2 (–3268C/ –2709T/–2661G/–2527A). In healthy individuals these SNPs are in linkage disequilibrium with the –2144 (GT)_n of the VWF promoter: haplotype 1 mainly segregates with short GT repeats (15–19, GTs), haplotype 2 with long repeats (GT ≧ 20, GT_L). Methods: We analyzed the (GT)_n locus, the SNP haplotypes and their association with VWF levels in 80 CS patients in order to precisely define the cortisol-sensitive VWF promoter pattern. CS patients were divided into groups A (increased VWF) and B (normal VWF). Results: Haplotype 1 and (GT)_S were more frequent in group A patients, and conferred a 9- and 7.5-fold risk of developing high VWF levels, respectively. Haplotype 2 and (GT)_L were more represented in group B. There was also an unexpected higher prevalence of recombinant SNP haplotypes in CS patients (6.2%) than in normals (0.9%), p = 0.002. Conclusions: Our results indicate that the cortisol-induced increase in VWF may be predicted by VWF promoter polymorphisms, haplotype 1 and (GT)_S being the sensitive pattern. These represent new markers for defining the prothrombotic risk of CS. The clinical significance, if any, of the increased recombination rate in SNP haplotypes in the VWF promoter warrants further study.

Functional characterization of a 13-bp deletion (c.-1522_-1510del13) in the promoter of the von Willebrand factor gene in type 1 von Willebrand disease

AbstractWe have studied the effect of a 13-bp deletion in the promoter of the von Willebrand factor (VWF) gene in a patient with type 1 von Willebrand disease. The index case has a VWF:Ag of 0.49 IU/mL and is heterozygous for the deletion. The deletion is located 48 bp 5′ of the transcription start site, and in silico analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies all predict aberrant binding of Ets transcription factors to the site of the deletion. Transduction of reporter gene constructs into blood outgrowth endothelial cells showed a 50.5% reduction in expression with the mutant promoter (n = 16, P < .001). A similar 40% loss of transactivation was documented in transduced HepG2 cells. A similar marked reduction of transgene expression was shown in the livers of mice injected with the mutant promoter construct (n = 8, P = .003). Finally, in studies of BOEC mRNA, the index case showed a 4.6-fold reduction of expression of the VWF transcript associated with the deletion mutation. These studies show that the 13-bp deletion mutation alters the binding of Ets (and possibly GATA) proteins to the VWF promoter and significantly reduces VWF expression, thus playing a central pathogenic role in the type 1 von Willebrand disease phenotype in the index case.

Endothelial histamine H 1 receptor signaling reduces blood–brain barrier permeability and susceptibility to autoimmune encephalomyelitis

Disruption of the blood-brain barrier (BBB) underlies the development of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis. Environmental factors, such as Bordetella pertussis, are thought to sensitize central endothelium to biogenic amines like histamine, thereby leading to increased BBB permeability. B. pertussis-induced histamine sensitization (Bphs) is a monogenic intermediate phenotype of EAE controlled by histamine H(1) receptor (Hrh1/H(1)R). Here, we transgenically overexpressed H(1)R in endothelial cells of Hrh1-KO (H(1)RKO) mice to test the role of endothelial H(1)R directly in Bphs and EAE. Unexpectedly, transgenic H(1)RKO mice expressing endothelial H(1)R under control of the von Willebrand factor promoter (H(1)RKO-vWF(H1R) Tg) were Bphs-resistant. Moreover, H(1)RKO-vWF(H1R) Tg mice exhibited decreased BBB permeability and enhanced protection from EAE compared with H(1)RKO mice. Thus, contrary to prevailing assumptions, our results show that endothelial H(1)R expression reduces BBB permeability, suggesting that endothelial H(1)R signaling may be important in the maintenance of cerebrovascular integrity.

Hypercortisolism and pregnancy upregulate von Willebrand factor through different mechanisms: report on a pregnant patient with Cushing's syndrome

Von Willebrand factor (VWF) is reportedly increased in pregnancy and Cushing's syndrome, inducing a hypercoagulable state. In Cushing's syndrome, VWF gene promoter polymorphisms modulate cortisol-dependent VWF upregulation, haplotype 1 (GCAG) and short GT-repeats (GT)(S) being the susceptible, and haplotype 2 (CTGA) and long GT-repeats (GT)(L) the protective pattern. We report on a Cushing's syndrome patient who became pregnant under hypercortisolism, in whom we monitored the evolution of her hypercoagulable state. During the active phase of Cushing's syndrome, the patient's VWF and factor VIII concentrations were normal, despite high urinary-free cortisol levels consistent with the presence of haplotype 2 and (GT)(L) alleles in the VWF gene promoter. VWF and factor VIII increased significantly and progressively after she became pregnant and peaked just before delivery, returning to normal 5 months later, while her hypercortisolism persisted. Our data indicate that two different mechanisms upregulate VWF under hypercortisolism and pregnancy, the latter being independent of the VWF promoter haplotypes sensitive to cortisol excess.

Repressors NFI and NFY Participate in Organ-Specific Regulation of von Willebrand Factor Promoter Activity in Transgenic Mice

To determine the role of repressors in cell type and organ-specific activation of von Willebrand factor (VWF) promoter sequences -487 to 247 in vivo. Activation patterns of wild-type and mutant VWF promoters (sequences -487 to 247) containing mutations in repressors nuclear factor-I (NFI)- and nuclear factor Y (NFY)-binding sites were analyzed in transgenic mice. Mutation of the NFI-binding site activated the promoter in heart and lung endothelial cells, whereas mutation of the NFY-binding site activated the promoter in kidney vasculature. Immunofluorescence analyses showed that NFIB was predominant in heart and lung endothelial cells, whereas NFIX was predominantly detected in kidney endothelial cell nuclei. By using chromatin immunoprecipitation, we demonstrated that the distal lung-specific enhancer (containing a YY1 site) of the VWF gene is brought in proximity to the NFI binding site. The NFI and NFY repressors contribute differentially to organ-specific regulation of the VWF promoter, and the organ-specific action of NFI may reflect its organ-specific isoform distribution. In addition, the lung-specific enhancer region of the endogenous VWF gene may inhibit NFI repressor function through chromatin looping, which can approximate the 2 regions.

Microsatellite (GT) n is part of the von Willebrand factor (VWF) promoter region that influences the glucocorticoid-induced increase in VWF in Cushing's syndrome

Introduction The cortisol-induced increase in von Willebrand factor (VWF) in Cushing's syndrome (CS) seems to depend on single nucleotide polymorphisms (SNPs) of the VWF promoter, haplotype 1 (-3268G/-2709C/-2661A/-2527G) being the susceptible pattern. Materials and Methods This study focused on a new variable region of the VWF promoter, the -2144(GT) n locus, to establish whether different GT-repeat lengths are also involved in modulating the cortisol-induced increase in VWF. Sixty-nine CS patients were investigated, divided into groups A (high VWF) and B (normal VWF). Results Analysing the (GT) n locus revealed a similar allele distribution in CS patients and normal subjects, (GT) n variants ranging from 15 to 24 repeats and (GT) 19 and (GT) 21 being the two most represented. However, when groups A and B were analysed separately, a different allele distribution was observed: short GT-repeats (15-19, GT S) were more frequent in group A, long GT-repeats (20-24, GT L) in group B (p = 0.01). About genotype distributions, (GT) S/(GT) S was higher in group A and rare in group B (22.5% and 3.4%, respectively), whereas (GT) L/(GT) L was higher in group B than in group A (55.2%, 27.5%) (p = 0.021). Odds-ratio analysis revealed a risk of a cortisol-dependent increase in VWF three times higher for alleles (GT) S than for (GT) L, and 13-fold for genotype (GT) S/(GT) S respect to (GT) L/(GT) L. Conclusions In conclusion, not only the SNPs haplotypes in the VWF gene promoter, but also the variable-length (GT) n locus predict the risk of developing high VWF levels under conditions of glucocorticoid excess; the combination of (GT) S and haplotype 1 represents the susceptible pattern.

A +220 GATA motif mediates basal but not endotoxin-repressible expression of the von Willebrand factor promoter in Hprt-targeted transgenic mice.

The von Willebrand factor (VWF) gene is a marker for spatial and temporal heterogeneity of the endothelium. A GATA motif at +220 has been implicated in basal VWF expression in vitro. Other studies have shown that GATA3 and VWF are transcriptionally downregulated in response to inflammatory mediators. Our goal was to determine the importance of the +220 GATA motif in mediating expression of VWF promoter in vivo, and to elucidate whether the GATA element plays a role in spatial and/or temporal regulation of VWF expression. ChIP and electrophoretic mobility shift assays were carried out in human umbilical vein endothelial cells (HUVEC). Reporter gene constructs containing 3.6 kb of the human VWF promoter with and without a mutation of the +220 GATA element were transfected into cultured endothelial cells or targeted to the Hprt locus of mice. The Hprt-targeted mice were subjected to endotoxemia. In protein-DNA binding assays, the +220 GATA motif bound GATA-2, -3 and -6. Mutation of the GATA site resulted in reduced basal promoter activity in HUVEC. When targeted to the Hprt locus of mice, the GATA mutation resulted in a significant, proportionate reduction of promoter activity in LacZ expressing vascular beds. Systemic administration of lipopolysaccharide (LPS) resulted in a widespread reduction in VWF mRNA expression and promoter activity. LPS-mediated repression of the VWF promoter was unaffected by the GATA mutation. A region of the VWF promoter between -2182 and the end of the first intron contains information for LPS-mediated gene repression. The +220 GATA motif is important for basal, but not LPS-repressible expression of the VWF gene.

Influence of a GT repeat element on shear stress responsiveness of the VWF gene promoter.

Plasma von Willebrand factor (VWF) is mainly derived from endothelial cells, cells that express a large repertoire of genes that are transcriptionally regulated by fluid shear stress. Endothelial VWF expression is not uniform throughout the vasculature, and levels are increased at regions associated with disturbed blood flow and steep gradients of shear stress. It is, however, unknown whether shear stress influences the regulation of VWF gene expression. Our objective was to evaluate the effect of shear stress on endogenous endothelial VWF mRNA expression and VWF promoter (-2722 to -1224) activity and to determine whether genetic elements modulate this flow-induced expression. A parallel plate flow chamber was used to expose endothelial cells to a shear level of 15 dynes cm(-2) for 24 or 6 h. VWF mRNA expression was analyzed. Various VWF promoter constructs that each contain either SNP haplotypes 1 or 2 and either a 17-GT or a 23-GT repeat element were transfected into endothelial cells, and flow-induced promoter activation was assessed. When endothelial cells were exposed to shear stress, endogenous VWF mRNA expression increased 1.84-fold and average VWF promoter activity was enhanced 3.4-fold. Single nucleotide polymorphisms at -2708 and -2525, and the shear stress-response element at -1585, are not responsible for the shear stress-induced increase. Rather a GT repeat element at -2124 mediates the increase in activity, and the length of this polymorphic repeat element influences the magnitude of induction. Shear stress enhances VWF promoter activity and a polymorphic GT repeat element mediates the stress-induced transactivation.