Sorting out the Weibel-Palade body

Abstract

Vascular endothelial cells form the inner lining of the vessel wall and thereby help maintaining the structural integrity of the blood vessel. In addition, the endothelium provides the surface for and actively participates in the mediation of inflammatory responses and in the arrest of bleeding in the event of vascular damage. Endothelial cells also take part in these processes by releasing the content of their unique, cigar-shaped organelles called Weibel-Palade bodies (WPBs). These organelles function as storage vesicles for von Willebrand factor (VWF), a multimeric adhesive glycoprotein crucial for platelet plug formation, the leukocyte receptor P-selectin and a number of bioactive compounds that include the chemokines interleukin-8 (IL-8) and eotaxin-3. To adequately respond to events like vascular trauma, endothelial cells are equipped with a complex signaling machinery that can regulate exocytosis of WPBs in response to cellular activation. The studies performed in this thesis are aimed at gaining a better understanding of the two processes that are fundamental to this capability of the endothelium, namely sorting of secretory proteins to WPBs and regulated secretion through exocytosis of WPBs. To gain more insight in how endothelial cells modulate the content of their WPBs, we have investigated a possible chaperone function for VWF by which it governs entry of other constituents into the storage pathway. Furthermore, this thesis aims to unravel signaling cascades and identify cellular components that control the exocytosis of WPBs in response to activation of the endothelial cell. In chapter 2, we have studied the entry of the chemokine IL-8 into the storage pathway of the endothelial cell in a quantitative manner. We have made the interesting observation that the molar amounts of IL-8 and VWF that enter WPBs are intimately linked. We postulate that this is due to a molecular interaction between these proteins in the acidic environment of the late secretory pathway. In chapter 3 we have found that the guanine exchange factor RalGDS regulates the release of WPBs in response to agonists that are mediated by the second messenger Ca2+. Through its interaction with the Ca2+ sensor CaM, RalGDS activates the small GTPase Ral in thrombin-stimulated endothelial cells, which results in the exocytosis of WPBs. Chapter 4 describes a novel PKA-independent pathway through which endothelial cells regulate cAMP-mediated exocytosis of WPB. This depends on the activation of the GTPase Rap1 by the cAMP-GEF Epac. In chapter 5 we have found that WPBs in endothelial cells that mimic a phenotype induced by shear stress display less clustering at the MTOC in response to stimulation with agonists that raise intracellular cAMP. Finally, in chapter 6 of this thesis our findings are summarized and we discuss them in the context of reports by others. A more general view is provided on the targeting of bioactive compounds to WPBs and regulatory mechanisms that exist in the endothelium to control release of these vesicles. Also, directions for future study are indicated.