Transcriptional regulation of von Willebrand Factor gene in response to hypoxia and in cancer cells

• Author / Creator

  Mojiri, Anahita

• Von Willebrand Factor (VWF) is a pro-coagulant, glycosylated protein that is exclusively expressed in endothelial cells and megakaryocytes. An acute phase protein, VWF is upregulated or released from activated or injured endothelial cells. In addition, it is a carrier for factor VIII that mediates the adhesion of platelets to the sub-endothelium at the injury sites of blood vessels. Both low and high levels of VWF protein may contribute to diseases associated with abnormal thrombosis. Low levels of VWF protein are the cause of the most prevalent inherited bleeding disorder, known as von Willebrand disease, while high levels of VWF protein, when dysregulated, are an independent risk factor for cardiovascular disease. Although VWF is a marker of endothelial cells, it exhibits a heterogeneic expression pattern throughout the vasculature. However, neither the molecular bases of VWF transcription, nor its heterogeneic expression pattern, nor the mechanisms of its response to external stimuli are completely known. Investigation of the VWF transcription process has led to the identification of distinct regions, as well as specific regulatory cis- and trans-acting factors, which contribute to this process. To gain more insight into the mechanisms that regulate VWF gene activity, we explored VWF expression in response to hypoxia in vivo and in vitro. We also explored de novo expression of VWF in cancer cells of non-endothelial origin. We investigated the VWF expression at protein and RNA levels in a mouse model of pulmonary hypertension that occurs as a result of hypoxia exposure. We demonstrated increased expression of VWF in all of the major organs of hypoxic compared to control mice. Furthermore, we specifically investigated the mechanisms underlying VWF upregulation in the hypoxic lung and heart. In the hypoxic lung, de novo expressions of VWF were detected in small microvessels, while in control mice, VWF expression was predominantly limited to large vessels of the lung. Hypoxia-induced upregulation of VWF in the lung endothelial cells was associated with increased binding of the YY1 transacting factor to its cognate binding site (intron 51 region), which was concomitant with increased translocation of YY1 to the nuclei. VWF upregulation in the lung was also associated with decreased binding of NFIB repressor to the promoter. We demonstrated that organ-specific mechanisms are involved in the hypoxia-induced regulation of VWF transcription. While hypoxia resulted in VWF upregulation in the heart and lungs, this was not observed in kidney endothelial cells either in vivo or in vitro. Furthermore, distinct mechanisms participated in the hypoxia-response of endothelial cells in the lung compared to those in the heart. This included a reduction in the binding of the repressor NFIB to the VWF promoter in both cell types. However, in lung endothelial cells, YY1 participated in the hypoxia-induction of VWF, while in cardiac endothelial cells, GATA6 and HIF participated in this process. Analysis of the methylation pattern demonstrated that a CpG dinucleotide located in the proximity of the repressor NFIB binding site was hypermethylated in response to hypoxia, specifically in cardiac endothelial cells. Increased VWF expression in the heart and lung of hypoxic mice was associated with platelet accumulation and aggregation in blood vessels, and occluded vessels were observed in the heart and lung. Increased plasma levels of VWF and thrombogenesis are commonly observed in cancer patients. It has been proposed that the source of these increased levels of VWF is either endothelial cells or platelets. However, we have demonstrated that a series of malignant glioma as well as osteosarcoma cell lines express VWF, which in turn, mediate increased cancer cell adhesion, transmigration and extravasation from endothelial cells. Analyses of the mechanisms of transcriptional activation of the VWF gene in cancer cell lines demonstrated a binding pattern of transacting factors and epigenetic modifications that was generally consistent with that observed in endothelial cells.

• Subjects / Keywords

  • VWF in Cancer
  • Gene regulation
  • Von Willebrand Factor

• Graduation date

  Fall 2016

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3ZG6GC68

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.