Summary The blood vessel wall has a number of self‐healing properties, enabling it to minimize blood loss and prevent or overcome infections in the event of vascular trauma. Endothelial cells prepackage a cocktail of hemostatic, inflammatory and angiogenic mediators in their unique secretory organelles, the Weibel–Palade bodies (WPBs), which can be immediately released on demand. Secretion of their contents into the vascular lumen through a process called exocytosis enables the endothelium to actively participate in the arrest of bleeding and to slow down and direct leukocytes to areas of inflammation. Owing to their remarkable elongated morphology and their secretory contents, which span the entire size spectrum of small chemokines all the way up to ultralarge von Willebrand factor multimers, WPBs constitute an ideal model system for studying the molecular mechanisms of secretory organelle biogenesis, exocytosis, and content expulsion. Recent studies have now shown that, during exocytosis, WPBs can undergo several distinct modes of fusion, and can utilize fundamentally different mechanisms to expel their contents. In this article, we discuss recent advances in our understanding of the composition of the WPB exocytotic machinery and how, because of its configuration, it is able to support WPB release in its various forms.

Supplemental Digital Content is available in the text.

Weibel-Palade bodies are endothelial secretory organelles that contain von Willebrand factor, P-selectin and CD63. Release of von Willebrand factor from Weibel-Palade bodies is crucial for platelet adhesion during primary hemostasis. Endosomal trafficking of proteins like CD63 to Weibel-Palade bodies during maturation is dependent on the adaptor protein complex 3 complex. Mutations in the AP3B1 gene, which encodes the adaptor protein complex 3 β1 subunit, result in Hermansky-Pudlak syndrome 2, a rare genetic disorder that leads to neutropenia and a mild bleeding diathesis. This is caused by abnormal granule formation in neutrophils and platelets due to defects in trafficking of cargo to secretory organelles. The impact of these defects on the secretory pathway of the endothelium is largely unknown. In this study, we investigated the role of adaptor protein complex 3-dependent mechanisms in trafficking of proteins during Weibel-Palade body maturation in endothelial cells. An ex vivo patient-derived endothelial model of Hermansky-Pudlak syndrome type 2 was established using blood outgrowth endothelial cells that were isolated from a patient with compound heterozygous mutations in AP3B1. Hermansky-Pudlak syndrome type 2 endothelial cells and CRISPR-Cas9-engineered AP3B1−/− endothelial cells contain Weibel-Palade bodies that are entirely devoid of CD63, indicative of disrupted endosomal trafficking. Hermansky-Pudlak syndrome type 2 endothelial cells have impaired Ca2+-mediated and cAMP-mediated exocytosis. Whole proteome analysis revealed that, apart from adaptor protein complex 3 β1, also the μ1 subunit and the v-SNARE VAMP8 were depleted. Stimulus-induced von Willebrand factor secretion was impaired in CRISPR-Cas9-engineered VAMP8−/−endothelial cells. Our data show that defects in adaptor protein complex 3-dependent maturation of Weibel-Palade bodies impairs exocytosis by affecting the recruitment of VAMP8.

BackgroundSynthesis of the hemostatic protein von Willebrand factor (VWF) drives formation of endothelial storage organelles called Weibel‐Palade bodies (WPBs). In the absence of VWF, angiogenic and inflammatory mediators that are costored in WPBs are subject to alternative trafficking routes. In patients with von Willebrand disease (VWD), partial or complete absence of VWF/WPBs may lead to additional bleeding complications, such as angiodysplasia. Studies addressing the role of VWF using VWD patient–derived blood outgrowth endothelial cells (BOECs) have reported conflicting results due to the intrinsic heterogeneity of patient‐derived BOECs.ObjectiveTo generate a VWF‐deficient endothelial cell model using clustered regularly interspaced short palindromic repeats (CRISPR) genome engineering of blood outgrowth endothelial cells.MethodsWe used CRISPR/CRISPR‐associated protein 9 editing in single‐donor cord blood–derived BOECs (cbBOECs) to generate clonal VWF −/− cbBOECs. Clones were selected using high‐throughput screening, VWF mutations were validated by sequencing, and cells were phenotypically characterized.ResultsTwo VWF −/− BOEC clones were obtained and were entirely devoid of WPBs, while their overall cell morphology was unaltered. Several WPB proteins, including CD63, syntaxin‐3 and the cargo proteins angiopoietin (Ang)‐2, interleukin (IL)‐6, and IL‐8 showed alternative trafficking and secretion in the absence of VWF. Interestingly, Ang‐2 was relocated to the cell periphery and colocalized with Tie‐2.ConclusionsCRISPR editing of VWF provides a robust method to create VWF‐ deficient BOECs that can be directly compared to their wild‐type counterparts. Results obtained with our model system confirmed alternative trafficking of several WPB proteins in the absence of VWF and support the theory that increased Ang‐2/Tie‐2 interaction contributes to angiogenic abnormalities in VWD patients.

Monomeric autotransporters have been extensively used for export of recombinant proteins to the cell surface of Gram-negative bacteria. A bottleneck in the biosynthesis of such constructs is the passage of the outer membrane, which is facilitated by the β-domain at the C terminus of an autotransporter in conjunction with the Bam complex in the outer membrane. We have evaluated eight β-domain constructs for their capacity to secrete fused proteins to the cell surface. These constructs derive from the monomeric autotransporters Hbp, IgA protease, Ag43 and EstA and the trimeric autotransporter Hia, which all were selected because they have been previously used for secretion of recombinant proteins. We fused three different protein domains to the eight β-domain constructs, being a Myc-tag, the Hbp passenger and a nanobody or VHH domain, and assessed expression, membrane insertion and surface exposure. Our results show that expression levels differed considerably between the constructs tested. The constructs that included the β-domains of Hbp and IgA protease appeared the most efficient and resulted in expression levels that were detectable on Coomassie-stained SDS-PAGE gels. The VHH domain appeared the most difficult fusion partner to export, probably due to its complex immunoglobulin-like structure with a tertiary structure stabilized by an intramolecular disulfide bond. Overall, the Hbp β-domain compared favorably in exporting the fused recombinant proteins, because it showed in every instance tested a good level of expression, stable membrane insertion and clear surface exposure.

During inflammation, endothelial cells are bombarded with cytokines and other stimuli from surrounding cells. Leukocyte extravasation and vascular leakage are both prominent but believed to be uncoupled as they occur in separate spatiotemporal patterns. In this study, we investigated a "double-hit" approach on primary human endothelial cells primed with LPS followed by histamine. Using neutrophil transendothelial migration (TEM) under physiological flow assays, we found that an LPS-primed endothelium synergistically enhanced neutrophil TEM when additionally treated with histamine, whereas the effects on neutrophil TEM of the individual stimuli were moderate to undetectable. Interestingly, the double-hit-induced TEM increase was not due to decreased endothelial barrier, increased adhesion molecule expression, or Weibel-Palade body release. Instead, we found that it was directly correlated with junctional remodeling. Compounds that increased junctional "linearity" (i.e., stability) counteracted the double-hit effect on neutrophil TEM. We conclude that a compound, in this case histamine (which has a short primary effect on vascular permeability), can have severe secondary effects on neutrophil TEM in combination with an inflammatory stimulus. This effect is due to synergic modifications of the endothelial cytoskeleton and junctional remodeling. Therefore, we hypothesize that junctional linearity is a better and more predictive readout than endothelial resistance for compounds aiming to attenuate inflammation.

No abstract

Vascular endothelial cells contain unique rod-shaped secretory granules, called Weibel-Palade bodies (WPBs), which contain a number of haemostatic, angiogenic and inflammatory mediators. Several components that are critical for regulated WPB exocytosis have been identified, including the small GTPase Rab27A and its effector synaptotagmin-like protein 4-a (Slp4-a), but the mechanism remains unclear. We have previously identified syntaxin binding protein 1 (STXBP1) as an endogenous Slp4-a binding partner involved in WPB release, along with the SNARE proteins syntaxin-2 and -3. In this study we investigated the possible role of syntaxin-2 and -3 in WPB exocytosis. We characterized the subcellular location of these syntaxins in endothelial cells using immunocytochemistry. Syntaxin-2 was primarily associated with the plasma membrane where it localized at VE-caderin-based adherens junctions and at integrin-based adhesions to the extracellular matrix. Interestingly, the t-SNARE syntaxin-3 was primarily associated with WPBs. To further explore its role in WPB biology we mapped the endothelial interaction partners of syntaxin-3 through an unbiased mass spectrometry approach using pull downs of lentivirally-expressed mEGFP-syntaxin-3 with anti-GFP nanobeads. Among its interaction partners are various SNAREs and associated proteins such as syntaxin binding proteins 2 and 5 (STXBP2/5), N-ethylmaleimide-sensitive factor (NSF), SNAP23 and α-SNAP, suggesting we successfully pulled down a SNARE complex and its regulatory machinery that are involved in exocytosis. We further addressed the role of syntaxin-3 in stimulated WPB exocytosis using siRNA-mediated knockdowns and found that depletion of syntaxin-3 led to a significant potentiation of Ca2+- as well as cAMP-mediated VWF secretion. In contrast, we observed a decrease in basal (unstimulated) VWF secretion after silencing of syntaxin-3, which amounts to a significantly increased intracellular VWF content. The potentiation of VWF secretion after depletion of syntaxin-3 was almost completely attributable to an increased WPB pool size; when corrected for increased WPB content the probability of stimulated release of a WPB was unaltered in the absence of syntaxin-3. Our data position syntaxin-3 as a WPB-linked SNARE-protein that regulates basal secretion of VWF. Disclosures No relevant conflicts of interest to declare.