Gatekeepers of the vasculature; regulation of exocytosis by syntaxin binding proteins. (LSBR 1244)
Project leader: Dr. ir. Ruben Bierings, Dept. of Plasma Proteins, Sanquin Research, Amsterdam
The endothelium forms a highly dynamic physical barrier that separates blood from the underlying tissues. It is critically involved in self-healing mechanisms of the blood vessel that prevent infection and excessive blood loss upon vascular injury. In the event of vascular damage, such as a wound, blood loss is minimized by immediate secretion of proteins that participate in the arrest of bleeding. These proteins, of which Von Willebrand factor (VWF) plays a critical role in the formation of a platelet plug, are released from typical subcellular organelles of the endothelium, so called Weibel-Palade bodies (WPBs). This process also releases inflammatory mediators that regulate adhesion and extravasation of leukocytes. Proper regulation of WPB release is of crucial importance for maintaining vascular homeostasis: failure to release (sufficient amounts of) VWF, as in Von Willebrand disease (VWD), leads to a bleeding tendency, while increased circulating levels of VWF are associated with higher risk of cardiovascular morbidity such as coronary heart disease, ischemic stroke and thrombosis. In this project we have studied the cellular processes that regulate exocytosis of WPBs, to gain more knowledge on the ability of our body to find a balance for secretion of VWF and how failure to do so results in bleeding and cardiovascular abnormalities.
During exocytosis proteins associated with the WPB are brought into contact with proteins found on the inside of the endothelial plasma membrane. Together these proteins form the SNARE complex, a molecular machine that catalyzes the fusion between the membrane of the WPB and the plasma membrane of the endothelial cell. Using an unbiased mass spectrometry screen for interactors of Slp4-a, a regulator of WPB exocytosis that we had previously identified, we found a number of novel regulatory components of the SNARE machinery, including syntaxin-2, syntaxin-3 and syntaxin-binding protein 1 (STXBP1). We found that endothelial cells that were depleted of STXBP1 with RNAi were severely impaired in their ability to release WPBs. Mutations in STXBP1 cause a rare but very severe epileptic disorder in babies and infants, early infantile epileptic encephalopathy 4 (EIEE4). This is caused by the inability to properly release neurotransmitters in the brain, a process that is also reliant on SNARE complexes. In collaboration with pediatric neurologists from the University of Antwerp (Belgium) we have isolated blood outgrowth endothelial cells (BOECs) from an EIEE4 patient. BOECs from the EIEE4 patient also have the mutation in STXBP1, leading to reduced levels of STXBP1, and also to impaired secretion of VWF. Interestingly, the levels of VWF found in the plasma of this EIEE4 patient were subnormal, possibly due to reduced exocytosis of WPBs. The results of this study have led to a better understanding of the molecular processes taking place during WPB exocytosis and shed light on how defects in this system can lead to vascular disease. This is important for rational design of new treatment and diagnostic strategies for patients with bleeding or cardiovascular abnormalities.