Docking and fusion sites in human endothelial cells imaged and measured by using atomic force microscopy

Abstract

The vascular endothelium with its salient location at the interface between blood and tissue plays a pivotal role in the process of blood coagulation and inflammation. The transition into a procoagulatory and proinflammatory state upon stimulation (i.e. neuropeptides) is referred to as endothelial cell activation. One fundamental characteristic of this activation is the induction of von Willebrand factor (vWF), IL‐8, and P‐selectin exocytosis. These molecules are stored in large (up to 3 µm) cone‐like vesicles called Weibel Palade bodies (WPBs). By using atomic force microscopy (AFM), we are able to visualize the apical surface topography of human endothelial cells with nanometer resolution. In addition, AFM allows to measure local cell stiffness with a spatial resolution of 100 nm. In previous studies, we showed that endothelial cells have a readily releasable pool of WPBs. In resting cells, this intracellular docked vesicle pool can be imaged as plasma membrane protrusions with a height of 140 ± 50 nm (±SEM; n = 8) and a diameter of 275 ± 85 nm (±SEM; n = 8). Stiffness measurements revealed that humps are characterized by decreased cell membrane stiffness of 30% compared to surrounding cell membrane due to a reduced subapical actin network. After stimulation of the cells with hyperosmolaric solutions or histamine, these docked WPBs immediately fuse with the plasma membrane forming large (diameter: approximately 500 nm) exocytotic pores and release vWF into the supernatant (measured by ELISA) and expose P‐selectin. Immunostaining of vWF was found to be localized next to the exocytotic pores imaged by AFM. The data indicate that human endothelial cells have a readily releasable pool of WPBs that allows the instantaneous release of vWF, IL‐8, and exposure of P‐selectin. These distinct areas of exocytosis are characterized by cell membrane protrusions and decreased cell membrane stiffness due to a reduced actin cortical network.