Aging Is Associated With Organ-Specific Alterations in the Level and Expression Pattern of von Willebrand Factor.

BACKGROUND: VWF (von Willebrand factor) is an endothelial-specific procoagulant protein with a major role in thrombosis. Aging is associated with increased circulating levels of VWF, which presents a risk factor for thrombus formation. METHODS: Circulating plasma, cellular protein, and mRNA levels of VWF were determined and compared in young and aged mice. Major organs were subjected to immunofluorescence analyses to determine the vascular pattern of VWF expression and the presence of platelet aggregates. An in vitro model of aging, using extended culture time of endothelial cells, was used to explore the mechanism of age-associated increased VWF levels. RESULTS: Increased circulating plasma levels of VWF with elevated levels of larger multimers, indicative of VWF functional activity, were observed in aged mice. VWF mRNA and cellular protein levels were significantly increased in the brains, lungs, and livers but not in the kidneys and hearts of aged mice. Higher proportion of small vessels in brains, lungs, and livers of aged mice exhibited VWF expression compared with young, and this was concomitant with increased platelet aggregate formation. Prolonged culture of endothelial cells resulted in increased cell senescence that correlated with increased VWF expression; VWF expression was specifically detected in senescent cultured endothelial cells and abolished in response to p53 knockdown. A significantly higher proportion of VWF expressing endothelial cells in vivo exhibited senescence markers SA-ß-Gal (senescence-associated ß-galactosidase) and p53 in aged mouse brains compared with that of the young. CONCLUSIONS: Aging elicits a heterogenic response in endothelial cells with regard to VWF expression, leading to organ-specific increase in VWF levels and alterations in vascular tree pattern of expression. This is concomitant with increased platelet aggregate formation. The age-associated increase in VWF expression may be modulated through the process of cell senescence, and p53 transcription factor contributes to its regulation.

In Silico Analysis to Explore Lineage-Independent and -Dependent Transcriptional Programs Associated with the Process of Endothelial and Neural Differentiation of Human Induced Pluripotent Stem Cells.

Despite a major interest in understanding how the endothelial cell phenotype is established, the underlying molecular basis of this process is not yet fully understood. We have previously reported the generation of induced pluripotent stem cells (iPS) from human umbilical vein endothelial cells and differentiation of the resulting HiPS back to endothelial cells (Ec-Diff), as well as neural (Nn-Diff) cell lineage that contained both neurons and astrocytes. Furthermore, the identities of these cell lineages were established by gene array analysis. Here, we explored the same arrays to gain insight into the gene alteration processes that accompany the establishment of endothelial vs. non-endothelial neural cell phenotypes. We compared the expression of genes that code for transcription factors and epigenetic regulators when HiPS is differentiated into these endothelial and non-endothelial lineages. Our in silico analyses have identified cohorts of genes that are similarly up- or downregulated in both lineages, as well as those that exhibit lineage-specific alterations. Based on these results, we propose that genes that are similarly altered in both lineages participate in priming the stem cell for differentiation in a lineage-independent manner, whereas those that are differentially altered in endothelial compared to neural cells participate in a lineage-specific differentiation process. Specific GATA family members and their cofactors and epigenetic regulators (DNMT3B, PRDM14, HELLS) with a major role in regulating DNA methylation were among participants in priming HiPS for lineage-independent differentiation. In addition, we identified distinct cohorts of transcription factors and epigenetic regulators whose alterations correlated specifically with the establishment of endothelial vs. non-endothelial neural lineages.

Age-Associated Increase in Thrombogenicity and Its Correlation with von Willebrand Factor.

Endothelial cells that cover the lumen of all blood vessels have the inherent capacity to express both pro and anticoagulant molecules. However, under normal physiological condition, they generally function to maintain a non-thrombogenic surface for unobstructed blood flow. In response to injury, certain stimuli, or as a result of dysfunction, endothelial cells release a highly adhesive procoagulant protein, von Willebrand factor (VWF), which plays a central role in formation of platelet aggregates and thrombus generation. Since VWF expression is highly restricted to endothelial cells, regulation of its levels is among the most important functions of endothelial cells for maintaining hemostasis. However, with aging, there is a significant increase in VWF levels, which is concomitant with a significant rise in thrombotic events. It is not yet clear why and how aging results in increased VWF levels. In this review, we have aimed to discuss the age-related increase in VWF, its potential mechanisms, and associated coagulopathies as probable consequences.

Regulation of von Willebrand Factor Gene in Endothelial Cells That Are Programmed to Pluripotency and Differentiated Back to Endothelial Cells.

Endothelial cells play a central role in physiological function and pathophysiology of blood vessels in health and disease. However, the molecular mechanism that establishes the endothelial phenotype, and contributes to its signature cell type-specific gene expression, is not yet understood. We studied the regulation of a highly endothelial-specific gene, von Willebrand factor (VWF), in induced pluripotent stem cells generated from primary endothelial cells (human umbilical vein endothelial cells [HUVEC] into a pluripotent state [HiPS]) and subsequently differentiated back into endothelial cells. This allowed us to explore how VWF expression is regulated when the endothelial phenotype is revoked (endothelial cells to HiPS), and re-established (HiPS back to endothelial cells [EC-Diff]). HiPS were generated from HUVECs, their pluripotency established, and then differentiated back to endothelial cells. We established phenotypic characteristics and robust angiogenic function of EC-Diff. Gene array analyses, VWF chromatin modifications, and transacting factors binding assays were performed on the three cell types (HUVEC, HiPS, and EC-Diff). The results demonstrated that generally cohorts of transacting factors that function as transcriptional activators, and those that contribute to histone acetylation and DNA demethylation, were significantly decreased in HiPS compared with HUVECs and EC-Diff. In contrast, there were significant increases in the gene expression levels of epigenetic modifiers that function as methyl transferases in HiPS compared with endothelial cells. The results demonstrated that alterations in chromatin modifications of the VWF gene, in addition to expression and binding of transacting factors that specifically function as activators, are responsible for establishing endothelial specific regulation of the VWF gene. Stem Cells 2019;37:542-554.

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

BACKGROUND AND AIMS: We 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. METHODS: We 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. RESULTS: Our 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. CONCLUSIONS: Heart and lung endothelial cells demonstrated VWF upregulation in response to hypoxia, using distinct mechanisms, while this response was lacking in kidney endothelial cells.

Von Willebrand factor contribution to pathophysiology outside of von Willebrand disease.

VWF is a procoagulant protein that plays a central role in the initiation of platelets aggregate formation and thrombosis. While von Willebrand disease has long been known to result from qualitative and quantitative deficiencies of VWF, it is recently that contribution of elevated levels of VWF to various pathological conditions including thrombosis, inflammation, angiogenesis, and cancer metastasis has been appreciated. Here, we discuss contribution of elevated levels of VWF to various thrombotic and nonthrombotic pathological conditions.

Functional assessment of von Willebrand factor expression by cancer cells of non-endothelial origin.

Von Willebrand factor (VWF) is a highly adhesive procoagulant molecule that mediates platelet adhesion to endothelial and subendothelial surfaces. Normally it is expressed exclusively in endothelial cells (ECs) and megakaryocytes. However, a few studies have reported VWF detection in cancer cells of non-endothelial origin, including osteosarcoma. A role for VWF in cancer metastasis has long been postulated but evidence supporting both pro- and anti-metastatic roles for VWF has been presented. We hypothesized that the role of VWF in cancer metastasis is influenced by its cellular origin and that cancer cell acquisition of VWF expression may contribute to enhanced metastatic potential. We demonstrated de novo expression of VWF in glioma as well as osteosarcoma cells. Endothelial monolayer adhesion, transmigration and extravasation capacities of VWF expressing cancer cells were shown to be enhanced compared to non-VWF expressing cells, and were significantly reduced as a result of VWF knock down. VWF expressing cancer cells were also detected in patient tumor samples of varying histologies. Analyses of the mechanism of transcriptional activation of the VWF in cancer cells demonstrated a pattern of trans-activating factor binding and epigenetic modifications consistent overall with that observed in ECs. These results demonstrate that cancer cells of non-endothelial origin can acquire de novo expression of VWF, which can enhance processes, including endothelial and platelet adhesion and extravasation, that contribute to cancer metastasis.

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.

Reprogramming of HUVECs into induced pluripotent stem cells (HiPSCs), generation and characterization of HiPSC-derived neurons and astrocytes.

Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling, drug development, screening, and the potential for "patient-matched" cellular therapies in neurodegenerative diseases. In this study, with the objective of establishing reliable tools to study neurodegenerative diseases, we reprogrammed human umbilical vein endothelial cells (HUVECs) into iPSCs (HiPSCs). Using a novel and direct approach, HiPSCs were differentiated into cells of central nervous system (CNS) lineage, including neuronal, astrocyte and glial cells, with high efficiency. HiPSCs expressed embryonic genes such as nanog, sox2 and Oct-3/4, and formed embryoid bodies that expressed markers of the 3 germ layers. Expression of endothelial-specific genes was not detected in HiPSCs at RNA or protein levels. HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons. These stem cell-derived neurons are susceptible to inflammatory cell-mediated neuronal injury. HiPSC-derived neurons express various amino acids that are important for normal function in the CNS. They have functional receptors for a variety of neurotransmitters such as glutamate and acetylcholine. HiPSC-derived astrocytes respond to ATP and acetylcholine by elevating cytosolic Ca2+ concentrations. In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes. These cells are appropriate tools for studying the development of the nervous system, the pathophysiology of various neurodegenerative diseases and the development of potential drugs for their treatments.

Plakoglobin interacts with the transcription factor p53 and regulates the expression of 14-3-3σ.

Plakoglobin (γ-catenin), a constituent of the adherens junction and desmosomes, has signaling capabilities typically associated with tumor/metastasis suppression through mechanisms that remain undefined. To determine the role of plakoglobin during tumorigenesis and metastasis, we expressed plakoglobin in human tongue squamous cell carcinoma (SCC9) cells and compared the mRNA profiles of parental SCC9 cells and their plakoglobin-expressing transfectants (SCC9-PG). We detected several p53-target genes whose levels were altered upon plakoglobin expression. In this study, we identified the p53 regulated tumor suppressor 14-3-3σ as a direct plakoglobin-p53 target gene. Coimmunoprecipitation experiments revealed that plakoglobin and p53 interact, and chromatin immunoprecipitation and electrophoretic mobility shift assays revealed that plakoglobin and p53 associate with the 14-3-3σ promoter. Furthermore, luciferase reporter assays showed that p53 transcriptional activity is increased in the presence of plakoglobin. Finally, knockdown of plakoglobin in MCF-7 cells followed by luciferase assays confirmed that p53 transcriptional activity is enhanced in the presence of plakoglobin. Our data suggest that plakoglobin regulates gene expression in conjunction with p53 and that plakoglobin may regulate p53 transcriptional activity, which may account, in part, for the tumor/metastasis suppressor activity of plakoglobin.

Hypoxia results in upregulation and de novo activation of von Willebrand factor expression in lung endothelial cells.

OBJECTIVE: 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. APPROACH AND RESULTS: 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. CONCLUSIONS: 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.

Repressors NFI and NFY participate in organ-specific regulation of von Willebrand factor promoter activity in transgenic mice.

OBJECTIVE: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

CLIC5A, a component of the ezrin-podocalyxin complex in glomeruli, is a determinant of podocyte integrity.

The chloride intracellular channel 5A (CLIC5A) protein, one of two isoforms produced by the CLIC5 gene, was isolated originally as part of a cytoskeletal protein complex containing ezrin from placental microvilli. Whether CLIC5A functions as a bona fide ion channel is controversial. We reported previously that a CLIC5 transcript is enriched approximately 800-fold in human renal glomeruli relative to most other tissues. Therefore, this study sought to explore CLIC5 expression and function in glomeruli. RT-PCR and Western blots show that CLIC5A is the predominant CLIC5 isoform expressed in glomeruli. Confocal immunofluorescence and immunogold electron microscopy reveal high levels of CLIC5A protein in glomerular endothelial cells and podocytes. In podocytes, CLIC5A localizes to the apical plasma membrane of foot processes, similar to the known distribution of podocalyxin and ezrin. Ezrin and podocalyxin colocalize with CLIC5A in glomeruli, and podocalyxin coimmunoprecipitates with CLIC5A from glomerular lysates. In glomeruli of jitterbug (jbg/jbg) mice, which lack the CLIC5A protein, ezrin and phospho-ERM levels in podocytes are markedly lower than in wild-type mice. Transmission electron microscopy reveals patchy broadening and effacement of podocyte foot processes as well as vacuolization of glomerular endothelial cells. These ultrastructural changes are associated with microalbuminuria at baseline and increased susceptibility to adriamycin-induced glomerular injury compared with wild-type mice. Together, the data suggest that CLIC5A is required for the development and/or maintenance of the proper glomerular endothelial cell and podocyte architecture. We postulate that the interaction between podocalyxin and subjacent filamentous actin, which requires ezrin, is compromised in podocytes of CLIC5A-deficient mice, leading to dysfunction under unfavorable genetic or environmental conditions.

Valproic acid exerts differential effects on CXCR4 expression in leukemic cells.

We recently reported that the histone deacetylase inhibitor, valproic acid (VPA), increases CXCR4 receptor expression and function in cord blood hematopoietic stem/progenitor cells (HSPC) and the immature, highly CD34-positive AML cell lines KG-1a and KG-1. In this study, we investigated whether VPA influences CXCR4 in CD34-negative AML cell lines (promyelocytic HL-60 and monocytic THP-1), as well as both CD34-positive and CD34-negative primary AML cells. We found that VPA (i) diminishes CXCR4 expression and chemotaxis in HL-60 cells and in the CD34-negative subtypes of primary AML cells and (ii) increases CXCR4 expression and function in the highly CD34-positive subtypes of primary AML cells. Hence, we suggest that VPA exerts different effects on CXCR4 depending on cell maturation status, and this novel finding may have important implications for AML therapy.

Valproic acid increases CXCR4 expression in hematopoietic stem/progenitor cells by chromatin remodeling.

A major limitation of cord blood (CB) hematopoietic stem/progenitor cell (HSPC) transplantation in adult patients is the low cell dose available, which is associated with delayed or failed engraftment. This has prompted intensive research to develop novel strategies to improve HSPC engraftment and reconstitution. The chemokine receptor CXCR4 and its ligand stromal cell-derived factor (SDF)-1alpha play a crucial role in the homing and repopulation capacity of HSPCs. We hypothesized that in HSPCs the CXCR4 receptor is regulated through chromatin remodeling by histone deacetylase inhibitors (HDIs) such as valproic acid (VPA). Using CB CD34(+) cells and the models of immature hematopoietic cells expressing CD34 antigen, namely the leukemic cell lines KG-1a and KG-1, we found that VPA increases surface and mRNA CXCR4 levels in these cells, thereby enhancing their migration toward an SDF-1alpha gradient. We also found that modulation of CXCR4 gene transcription by VPA correlates with the acetylation status of histone H4 in CB CD34(+) and KG-1 cells. Hence we suggest that in CB transplantation priming of HSPCs with VPA could improve homing and engraftment.

Sequences in intron 51 of the von Willebrand factor gene target promoter activation to a subset of lung endothelial cells in transgenic mice.

In vivo analyses of the VWF promoter previously demonstrated that a fragment spanning sequences -487 to +247 targets promoter activation to brain vascular endothelial cells, whereas a longer fragment including 2182 bp of the 5'-flanking sequences, the first exon, and the first intron activated expression in endothelial cells of the heart and muscles as well as the brain of transgenic mice. These results suggested that additional VWF gene sequences were required for expression in other vascular endothelial cells in vivo. We have now identified a region within intron 51 of the VWF gene that is DNase I-hypersensitive (HSS) specifically in non-endothelial cells and interacts with endothelial and non-endothelial specific complexes that contain YY1. We demonstrate that beta-actin is associated with YY1 specifically in the nucleus of non-endothelial cells and is a component of the nuclear protein complexes that interact with the DNase I-hypersensitive region. In vitro transfection analyses demonstrated that HSS sequences containing this YY1-binding site do not significantly affect VWF promoter activity. However, in vivo analyses demonstrated that addition of these sequences to the VWF promoter (-487 to +247) results in promoter activation in lung and brain vascular endothelial cells. These results demonstrate that the HSS sequences in intron 51 of the VWF gene contain cis-acting elements that are necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo.

Histone H1-like protein participates in endothelial cell-specific activation of the von Willebrand factor promoter.

A region of the von Willebrand factor (VWF) promoter has been identified that is necessary to confer endothelial cell-specific activation to the VWF promoter. This region spans sequences +155 to +247 and contains binding sites for GATA6 and NFY transcription factors. To identify potential DNA binding transcription factors that directly interact with these sequences in an endothelial-specific manner, we have performed extensive gel mobility assays with use of 7 overlapping DNA probes that collectively span this entire region. An endothelial-specific protein DNA complex was formed with an oligonucleotide that corresponded to sequences +155 to +184 of the VWF gene. Mutation analysis identified a 6-nucleotide element corresponding to sequences +164 to +169 as the core-binding region for the formation of this complex. Transfection analysis demonstrated that the mutation, which abolished DNA-protein interaction, resulted in significant inhibition of the VWF promoter activity. DNA pull-down analysis, mass spectrometry, and Western blot analysis demonstrated that a 32-kDa polypeptide with homology to histone H1 constituted the endothelial-specific DNA binding protein, or a DNA binding subunit of this protein complex. On the basis of these results, we hypothesize that an H1-like protein functions as an endothelial cell-specific transcriptional activator of the VWF promoter.

Von Willebrand factor promoter targets the expression of amyloid beta protein precursor to brain vascular endothelial cells of transgenic mice.

Dysfunction of brain vascular endothelial cells may be associated with the pathogenesis of several diseases including cerebral amyloid angiopathy, hemorrhagic stroke and Alzheimer disease. New model systems are necessary to examine the contribution of brain endothelial cells in these disorders. The Von Willebrand factor gene promoter fragment that spans sequences -487 to +247 targets the expression of LacZ marker gene in transgenic mice specifically to brain vascular endothelial cells. Transgenic mice have been prepared that express human amyloid beta protein precursor protein (AbetaPP) isoforms 695 and 751 (wild-type and Dutch variant mutations) under the regulation of this VWF promoter sequence. These AbetaPP transgenes are specifically expressed in brain vascular endothelial cells. The VWF promoter is a valuable tool for targeting gene expression to brain vascular endothelial cells to provide a model to directly examine endothelial cell placement of genes and their contribution to cerebral vascular disease.

The NFY transcription factor inhibits von Willebrand factor promoter activation in non-endothelial cells through recruitment of histone deacetylases.

Human von Willebrand factor (VWF) gene sequences +155 to +247 contain cis-acting elements that contribute toward endothelial specific activation of the VWF promoter. Analyses of this region demonstrated the presence of a GATA-binding site that is necessary for the promoter activation in endothelial cells. We have reported recently the presence of a novel NFY-binding sequence in this region that does not conform to the consensus NFY-binding sequence CCAAT. NFY was shown to function as a repressor of the VWF promoter through interaction with this novel binding site. Here we report that the NFY interacts with histone deacetylases (HDACs) in a cell type-specific manner and recruits them to the VWF promoter to inhibit the promoter activity in non-endothelial cells. Analyses of the acetylation status of histones in the chromatin region containing the VWF promoter sequences demonstrated that these sequences are associated with acetylated histone H4 specifically in endothelial cells. It was also demonstrated that HDACs are specifically recruited to the same chromatin region in non-endothelial cells. We also demonstrated that GATA6 is the GATA family member that interacts with the VWF promoter and that GATA6 is associated with NFY specifically in non-endothelial cells. We propose that NFY recruits HDACs to the VWF promoter, which may result in deacetylation of GATA6 as well as of histones in non-endothelial cells, thus leading to promoter inactivation. In endothelial cells, however, association of HDACs, NFY, and GATA6 is interrupted potentially through endothelial cell-specific signaling/mechanism, thus favoring the balance toward acetylation and activation of the VWF promoter.

The NFY transcription factor functions as a repressor and activator of the von Willebrand factor promoter.

Human von Willebrand factor (VWF) gene sequences -487 to +247 function as an endothelial-specific promoter in vitro. Analysis of the activation mechanism of the VWF promoter has resulted in the identification of a number of cis-acting elements and trans-acting factors that regulate its activity. The GATA and Ets transcription factors were shown to function as activators of transcription, whereas NF1 and Oct1 were shown to repress transcription. We have reported the presence of another repressor element in exon 1 that interacted with a protein complex designated "R." In the absence of NF1 binding, inhibition of this interaction resulted in promoter activation in nonendothelial cells. We have now identified the "R" protein complex as the NFY transcription factor. Using DNA methylation interference assay and base substitution mutation analysis, we show that NFY interacts with a novel DNA sequence corresponding to nucleotides +226 to +234 in the VWF promoter that does not conform to the consensus NFY binding sequence CCAAT. The VWF gene does contain a CCAAT element that is located downstream of the TATA box and we show that the NFY factor also interacts with this CCAAT element. Using antibodies specific against the A, B, and C subunits of NFY, we demonstrate that the NFY complexes interacting with the CCAAT sequence have a composition similar to that of the repressor binding to the first exon sequences. The results of mutation analysis and transfection studies demonstrated that the interaction of NFY with the upstream CCAAT element is required for VWF promoter activation. Based on these results, we hypothesize that NFY can function both as a repressor and activator of transcription and its function may be modulated through its DNA binding sequences.