Abstract
Vascular endothelial cells line the luminal surface of the vasculature and form a barrier between circulating blood and the underlying tissues. The endothelium is able to respond to environmental signals, such as vascular injury or inflammation, by release of biologically active molecules. To this purpose, the endothelial cells contain different
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types of granules, including Weibel-Palade bodies (WPBs), unique storage organelles for the haemostatic glycoprotein von Willebrand Factor (VWF). WPBs release their content upon stimulation of endothelial cells with a large number of secretagogues like thrombin, epinephrine, vasopressin, histamine and forskolin. In addition to the primary WPB component VWF, a number of additional proteins with various functions are known to be recruited to these organelles, including P-selectin, angiopoietin-2 (Ang2) and interleukin-8 (IL-8). This thesis describes the results of a number of studies on the regulation of the content of WPBs as well as on the identification of novel WPB components.
Endothelial cells in unbranched segments of the vascular tree are subjected to blood flow-derived shear stress. The effect of the shear-stress induced transcription factor Krüppel-like factor 2 (KLF2) on WPBs was studied in Chapter 2. We observed changes in amount, dynamics and pro-inflammatory content of WPBs upon expression of KLF2 in endothelial cells. These results suggest that KLF2-expressing endothelium is less prone to vascular remodeling and inflammation.
In Chapter 3, a novel WPB component, insulin-like growth factor binding protein 7 (IGFBP7), was identified using a proteomics approach. Targeting of IGFBP7 to pseudo-WPBs in HEK293 cells was shown to be dependent on the carboxy-terminal D4-C1-C2-C3-CK domains of VWF. IGFBP7 was found to bind to ultralarge VWF strings that are released upon exocytosis of WPBs under flow.
In Chapter 4, a different proteomics approach was used to quantitatively analyze the secretome of agonist-induced endothelial cells. 179 proteins were found to be enriched in the secretome of agonist-induced cells, including secreted protein acidic and rich in cysteines (SPARC), which was found to colocalize with VWF in small round vesicles in endothelial cells. Immunocytochemistry studies suggest that the identified spherical vesicles are either constitutive vesicles or represent a population of vesicles that buds off during WPB maturation.
In Chapter 5, a new WPB component, angiopoietin-like protein 1 (Angptl1), was identified. We showed that targeting of both Angptl1 and Ang2 to WPBs is dependent on the FReD domain of these proteins. The D1-D3 domains of VWF appeared to be involved in targeting of both Angptl1 and Ang2 to pseudo-WPBs in HEK293 cells. These results suggest that a similar sorting mechanism might be used for targeting of Ang2 and Angptl1 to WPBs.
In the final chapter, Chapter 6, the results of the studies described in this thesis and their implications are discussed with reference to findings reported by other investigators
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