ENDOTHELIAL CELL HETEROGENEITY: A CORRELATION WITH THE VON WILLEBRAND FACTOR EXPRESSION IN DISTINCT ORGANS

• Author / Creator

  Lorenzana Carrillo, Maria A

• Endothelial cells (EC) of different organs exhibit heterogeneity in structure, function and gene expression pattern. This also extends to the pattern in which the highly endothelial-specific gene, von Willebrand Factor (VWF) is expressed. VWF is a multimeric adhesive, pro-coagulant glycoprotein involved in regulation of hemostasis and thrombosis; this protein is exclusively expressed in endothelial cells and megakaryocytes. Its two primary functions are to mediate the adhesion of platelets to the underlying endothelium and to be a carrier for the coagulation factor VIII. Deficiency in quantity and quality of the VWF protein is the cause of the most common inherited bleeding disorder, the von Willebrand Disease (VWD); on the other hand, dysregulated high levels increase the risk of thrombosis and cardiovascular disease. It was previously demonstrated that nucleotides -487 to +247 of the VWF gene function as an endothelial-specific promoter that exhibits organ-specific activity. We hypothesized that pattern of expression of transcription factors that regulate the VWF promoter may contribute to the mechanism that governs the organ-specific regulation of the VWF promoter. VWF promoter contains a binding site for GATA family of transacting factors and mutation of this site was shown to abolish the VWF promoter activity in vitro and in vivo. Several members of GATA family, including GATA2, 3 and 6 were reported to interact with the VWF promoter. We tested the hypothesis that there may be organ-specific distribution of GATA family members, which could contribute to organ-specific regulation of VWF. Immunofluorescence staining was used in various murine organs to mark the VWF and CD31 expressing EC. We also co-stained for transacting factor GATA family members GATA2, 3 and 6. Laser Capture Microdissection (LCM) was used to mark and dissect lung vessels’ EC that expressed VWF. RT-PCR was used to analyze gene expression pattern in the dissected cells. Our IF and confocal microscopy demonstrated that ECs of distinct organs exhibit distinct patterns of GATA isoforms. Using IF and LCM we could positively identify, capture and isolate target cells. RT-PCR analyses of isolated target cells demonstrated significant VWF expression in dissected EC, which was comparable to cultured EC. These data suggest that we have an organ-specific expression pattern of GATA transcription factors that may participate in the organ-specific regulation of VWF transcription. In order to gain more insight into the mechanisms that regulate VWF gene activity in vivo, I contributed to the exploration of how endothelial cells of different organs regulate VWF transcription in response to different stimuli, such as aging and hypoxia. The use of laser capture microdissection system will allow us to specifically detect the expression pattern of distinct regulatory factors that participate in the regulation of the VWF gene in EC of distinct organs not only in normal conditions but also in pathophysiological conditions. I was also involved in the project that investigates the expression of VWF in cancer cells of non-endothelial origin, since determining the mechanism of acquired VWF transcription by these cells would provide new insights towards understanding which VWF regulatory elements play a dominant role in establishing this activation.

• Subjects / Keywords

  • Endothelial Cells
  • Von Willebrand Factor
  • Endothelial Heterogeneity

• Graduation date

  Spring 2017

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3H41K028

• 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.