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http://hdl.handle.net/11375/28183
Title: | The Characterization of VWF in Mouse and Human Endothelial Cells |
Authors: | Kodeeswaran, Arthane |
Advisor: | Matino, Davide |
Department: | Medical Sciences |
Publication Date: | 2022 |
Abstract: | Von Willebrand factor (VWF) is a protein that secreted by endothelial cells and Weibel-Palade bodies of endothelial cells that mediates platelet adhesion and leads to the subsequent clotting at the site of injury. VWF plays a role in hemostatic diseases such as thrombosis. Due to its role in disease, it may be a novel target for the treatment of these diseases. RNA interference (RNAi) is a natural defence mechanism that is being investigated as a therapy for its ability to provide a precise and personalized treatment by silencing specific genes and preventing the translation of target proteins. This makes gene silencing promising for the treatment for many diseases, and VWF knockdown through siRNA could be a potential new strategy for VWF-associated disease. Current methods of in vitro study of VWF and ultimately VWD, comprise of the use of VWF-transfected heterologous cells or endothelial cells; however, there are no comprehensive studies investigating the regulation and secretion of VWF over time by endothelial cells, specifically murine endothelial cells that best translate in vivo conditions. Any current studies on VWF expression over time are at a shorter time scale that is not compatible with in vitro siRNA transfection protocols. To design siRNA transfection experiments to knockdown VWF in vitro, it is necessary to first investigate VWF expression in human and mouse endothelial cells at time points that can be applied to siRNA transfection. To investigate this aim, several cell lines (HUVEC, LSEC, LMEC and iMAEC) were grown in 24-well plates and treated with PMA, thrombin, or left untreated. RNA, protein lysates and supernatant samples were collected 48 hours post-treatment. Media was changed at 48h and 30-minute, 6h and 24h samples were collected after the media change for analysis through ELISA and qRT-PCR. VWF expression was also visualized in these cells using immunofluorescence. Our findings demonstrate that the expression of VWF in human versus mouse endothelial cells are different, with most similarities demonstrated between HUVECs and LSEC with the expected increase in VWF demonstrated at 48 hours. This data can be used to choose a cell line ideal for future experiments with a longer time scale and/or alter experimental designs depending on the cell line used. |
URI: | http://hdl.handle.net/11375/28183 |
Appears in Collections: | Open Access Dissertations and Theses |
Files in This Item:
File | Description | Size | Format | |
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Kodeeswaran_Arthane_finalsubmission202212_MSc.pdf | 1.23 MB | Adobe PDF | View/Open |
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