Spire1 and Myosin Vc promote Ca2+-evoked externalization of von Willebrand factor in endothelial cells.

Weibel-Palade bodies (WPB) are endothelial cell-specific storage granules that regulate vascular hemostasis by releasing the platelet adhesion receptor von Willebrand factor (VWF) following stimulation. Fusion of WPB with the plasma membrane is accompanied by the formation of actin rings or coats that support the expulsion of large multimeric VWF fibers. However, factor(s) organizing these actin ring structures have remained elusive. We now identify the actin-binding proteins Spire1 and Myosin Vc (MyoVc) as cytosolic factors that associate with WPB and are involved in actin ring formation at WPB-plasma membrane fusion sites. We show that both, Spire1 and MyoVc localize only to mature WPB and that upon Ca2+ evoked exocytosis of WPB, Spire1 and MyoVc together with F-actin concentrate in ring-like structures at the fusion sites. Depletion of Spire1 or MyoVc reduces the number of these actin rings and decreases the amount of VWF externalized to the cell surface after histamine stimulation.

Targeting Procalcitonin Protects Vascular Barrier Integrity.

Rationale: Capillary leakage frequently occurs during sepsis and after major surgery and is associated with microvascular dysfunction and adverse outcome. Procalcitonin is a well-established biomarker in inflammation without known impact on vascular integrity. Objectives: We determined how procalcitonin induces endothelial hyperpermeability and how targeting procalcitonin protects vascular barrier integrity. Methods: In a prospective observational clinical study, procalcitonin levels were assessed in 50 patients who underwent cardiac surgery and correlated to postoperative fluid and vasopressor requirements along with sublingual microvascular functionality. Effects of the procalcitonin signaling pathway on endothelial barrier and adherens junctional integrity were characterized in vitro and verified in mice. Inhibition of procalcitonin activation by dipeptidyl-peptidase 4 (DPP4) was evaluated in murine polymicrobial sepsis and clinically verified in cardiac surgery patients chronically taking the DPP4 inhibitor sitagliptin. Measurements and Main Results: Elevated postoperative procalcitonin levels identified patients with 2-fold increased fluid requirements (P < 0.01), 1.8-fold higher vasopressor demand (P < 0.05), and compromised microcirculation (reduction to 63.5 ± 2.8% of perfused vessels, P < 0.05). Procalcitonin induced 1.4-fold endothelial and 2.3-fold pulmonary capillary permeability (both Ps < 0.001) by destabilizing VE-cadherin. Procalcitonin effects were dependent on activation by DPP4, and targeting the procalcitonin receptor or DPP4 during sepsis-induced hyperprocalcitonemia reduced capillary leakage by 54 ± 10.1% and 60.4 ± 6.9% (both Ps < 0.01), respectively. Sitagliptin before cardiac surgery was associated with augmented microcirculation (74.1 ± 1.7% vs. 68.6 ± 1.9% perfused vessels in non-sitagliptin-medicated patients, P < 0.05) and with 2.3-fold decreased fluid (P < 0.05) and 1.8-fold reduced vasopressor demand postoperatively (P < 0.05). Conclusions: Targeting procalcitonin's action on the endothelium is a feasible means to preserve vascular integrity during systemic inflammation associated with hyperprocalcitonemia.

Proximity proteomics of endothelial Weibel-Palade bodies identifies novel regulator of von Willebrand factor secretion.

Weibel-Palade bodies (WPB) are unique secretory organelles of endothelial cells that store factors regulating vascular hemostasis and local inflammation. Endothelial activation triggers rapid exocytosis of WPB, leading to the surface presentation of adhesion molecules relevant for leukocyte rolling (P-selectin) and platelet capture (von Willebrand factor [VWF]). Despite its role as an important secretory organelle, a comprehensive compilation of factors associated with WPB has not been carried out. We addressed this via a proximity proteomics approach employing the peroxidase APEX2 coupled with 2 known WPB-associated proteins: the Rab GTPases Rab3b and Rab27a. We show that APEX2-Rab3b/27a fusion constructs are correctly targeted to WPB of primary endothelial cells, and that proteins in their close proximity can be biotinylated through the WPB-recruited APEX2. Mass spectrometry analysis of the biotinylated proteins identified 183 WPB-associated proteins. Whereas these include factors reported before to localize to WPB, the majority comprises proteins not previously associated with WPB biology. Among them, the SNARE-interacting protein Munc13-2 was shown here to specifically localize to WPB and to serve as a novel factor promoting histamine-evoked WPB exocytosis and VWF secretion. Thus, APEX2-based proximity proteomics can be used to specifically identify novel organelle-associated factors in primary endothelial cells.

Regulation of von-Willebrand Factor Secretion from Endothelial Cells by the Annexin A2-S100A10 Complex.

Endothelial cells serve as gatekeepers of vascular hemostasis and local inflammatory reactions. They can rapidly respond to changes in the environment, caused, for example, by blood vessel injury, tissue damage or infection, by secreting in a strictly regulated manner factors regulating these processes. These factors include adhesion receptors for circulating leukocytes and platelets, P-selectin and von-Willebrand factor (VWF) that are stored in specialized secretory granules of endothelial cells, the Weibel-Palade bodies (WPB). Acute exposure of these adhesion molecules converts the endothelial cell surface from an anti-adhesive state enabling unrestricted flow of circulating blood cells to an adhesive one capable of capturing leukocytes (through P-selectin) and platelets (through VWF). While these are important (patho)physiological responses, compromised or dysregulated WPB secretion can cause pathologies such as excessive bleeding or vascular occlusion. Several factors are involved in regulating the exocytosis of WPB and thus represent potential targets for therapeutic interventions in these pathologies. Among them, the annexin A2 (AnxA2)-S100A10 complex has been shown to participate in the tethering/docking of secretion-competent WPB at the plasma membrane, and interference with AnxA2/S100A10 expression or complex formation significantly reduces acute WPB exocytosis and VWF release. Thus, developing specific means to efficiently block AnxA2-S100A10 complex formation in endothelial cells could lead to novel avenues towards interfering with acute vascular thrombosis.

Analysis of Ca2+-Dependent Weibel-Palade Body Tethering by Live Cell TIRF Microscopy: Involvement of a Munc13-4/S100A10/Annexin A2 Complex.

Endothelial cells respond to blood vessel injury by the acute release of the procoagulant von Willebrand factor, which is stored in unique secretory granules called Weibel-Palade bodies (WPBs). Stimulated, Ca2+-dependent exocytosis of WPBs critically depends on their proper targeting to the plasma membrane, but the mechanism of WPB-plasma membrane tethering prior to fusion is not well characterized. Here we describe a method to visualize and analyze WPB tethering and fusion in living human umbilical vein endothelial cells (HUVEC) by total internal reflection fluorescence (TIRF) microscopy. This method is based on automated object detection and allowed us to identify components of the tethering complex of WPBs and to monitor their dynamics in space and time. An important tethering factor identified by this means was Munc13-4 that was shown to interact with S100A10 residing in a complex with plasma membrane-bound annexin A2.

Phosphorescent cationic iridium(iii) complexes dynamically bound to cyclodextrin vesicles: applications in live cell imaging.

We report cationic Ir(iii) complexes functionalized with adamantyl groups designed to bind to ß-cyclodextrin vesicles (CDV) with high affinity (K a = 1 × 106 M-1). The emission of the complexes is tuned by changing the nature of the cyclometalating ligands. The host-guest adduct of CDV and Ir(iii) complexes shows increased and significantly blue-shifted emission due to the lower mobility of the Ir(iii)-complexes residing in the less polar environment of the vesicle surface. Ir(iii)-decorated CDV are efficiently taken up by cells and can be used in live cell imaging. The CDV act as carriers to transport the phosphorescent complexes into cells where they selectively stain mitochondria.

A novel Munc13-4/S100A10/annexin A2 complex promotes Weibel-Palade body exocytosis in endothelial cells.

Endothelial cells respond to blood vessel injury by the acute release of the procoagulant von Willebrand factor, which is stored in unique secretory granules called Weibel-Palade bodies (WPBs). Stimulated WPB exocytosis critically depends on their proper recruitment to the plasma membrane, but factors involved in WPB-plasma membrane tethering are not known. Here we identify Munc13-4, a protein mutated in familial hemophagocytic lymphohistiocytosis 3, as a WPB-tethering factor. Munc13-4 promotes histamine-evoked WPB exocytosis and is present on WPBs, and secretagogue stimulation triggers an increased recruitment of Munc13-4 to WPBs and a clustering of Munc13-4 at sites of WPB-plasma membrane contact. We also identify the S100A10 subunit of the annexin A2 (AnxA2)-S100A10 protein complex as a novel Munc13-4 interactor and show that AnxA2-S100A10 participates in recruiting Munc13-4 to WPB fusion sites. These findings indicate that Munc13-4 supports acute WPB exocytosis by tethering WPBs to the plasma membrane via AnxA2-S100A10.