Weibel-Palade Bodies Research Articles

Interaction networks of Weibel-Palade body regulators syntaxin-3 and syntaxin binding protein 5 in endothelial cells

The endothelium stores the hemostatic protein Von Willebrand factor (VWF) in endothelial storage organelles called Weibel-Palade bodies (WPBs). During maturation, WPBs recruit a complex of Rab GTPases and effectors that associate with components of the SNARE machinery that control WPB exocytosis. Recent genome wide association studies have found links between genetic variations in the SNAREs syntaxin-2 (STX2) and syntaxin binding protein 5 (STXBP5) and VWF plasma levels, suggesting a role for SNARE proteins in regulating VWF release. Moreover, we have previously identified the SNARE proteins syntaxin-3 and STXBP1 as regulators of WPB release. In this study we used an unbiased iterative interactomic approach to identify new components of the WPB exocytotic machinery. An interactome screen of syntaxin-3 identifies a number of SNAREs and SNARE associated proteins (STXBP2, STXBP5, SNAP23, NAPA and NSF). We show that the VAMP-like domain (VLD) of STXBP5 is indispensable for the interaction with SNARE proteins and this capacity of the VLD could be exploited to identify an extended set of novel endothelial SNARE interactors of STXBP5. In addition, an STXBP5 variant with an N436S substitution, which is linked to lower VWF plasma levels, does not show a difference in interactome when compared with WT STXBP5. SignificanceThe hemostatic protein Von Willebrand factor plays a pivotal role in vascular health: quantitative or qualitative deficiencies of VWF can lead to bleeding, while elevated levels of VWF are associated with increased risk of thrombosis. Tight regulation of VWF secretion from WPBs is therefore essential to maintain vascular homeostasis. We used an unbiased proteomic screen to identify new components of the regulatory machinery that controls WPB exocytosis. Our data expand the endothelial SNARE protein network and provide a set of novel candidate WPB regulators that may contribute to regulation of VWF plasma levels and vascular health.

Cancer stem cells contribute to angiogenesis and lymphangiogenesis in serous adenocarcinoma of the ovary.

The origin of blood and lymphatic vessels in high-grade serous adenocarcinoma of ovary (HGSOC) is uncertain. We evaluated the potential of cancer stem cells (CSCs) in HGSOC to contribute to their formation. Using spheroids as an in vitro model for CSCs, we have evaluated their role in primary malignant cells (PMCs) in ascites from previously untreated patients with HGSOC and cell lines. Spheroids from PMCs grown under specific conditions showed significantly higher expression of endothelial, pericyte and lymphatic endothelial markers. These endothelial and lymphatic cells formed tube-like structures, showed uptake of Dil-ac-LDL and expressed endothelial nitric oxide synthase confirming their endothelial phenotype. Electron microscopy demonstrated classical Weibel-Palade bodies in differentiated cells. Genetically, CSCs and the differentiated cells had a similar identity. Lineage tracking using green fluorescent protein transfected cancer cells in nude mice confirmed that spheroids grown in stem cell conditions can give rise to all three cells. Bevacizumab, a monoclonal antibody that targets vascular endothelial growth factor inhibited the differentiation of spheroids to endothelial cells in vitro. These results suggest that CSCs contribute to angiogenesis and lymphangiogenesis in serous adenocarcinoma of the ovary, which can be inhibited.

PF804 SEC22B‐DEPENDENT TRAFFICKING IS ESSENTIAL FOR VWF MULTIMERIZATION AND FORMATION OF ELONGATED WPBS

Background:Von Willebrand factor (VWF) is a key hemostatic glycoprotein that is predominantly synthesized in endothelial cells. Upon synthesis, the pro‐VWF precursor dimerizes in the ER and is subsequently transported to the Golgi where it undergoes proteolytical processing, multimerization and finally is stored in newly emerging secretory organelles, known as Weibel‐Palade bodies (WPBs). Assembly of VWF into high molecular weight (HMW) multimers, packaging in elongated WPBs and regulated release to the extracellular environment as ultra‐long VWF strings is crucial for its ability to recruit platelets to sites of vascular injury. The adhesive properties of VWF are strongly tied to the length of the secretory organelles in which it's contained in, however, how endothelial cells control the dimensions of their WPBs is still unclear.Aims:In this study, we investigated the crucial role of SNARE‐mediated ER/Golgi trafficking in VWF maturation and WPB biogenesis.Methods:In a shRNA screen of longin‐containing SNAREs in primary endothelial cells, we identified the proteinsinvolved in ER/Golgi trafficking. Protein expression was assessed by Western Blot and the morphology of intracellular compartment was studied using confocal microscopy.Results:We found that silencing of Sec22b resulted in the loss of the typically elongated morphology of WPBs. VWF was partially retained in the ER, resulting in a significantly higher ratio of proVWF:VWF. Decreased VWF influx to the Golgi affected the morphology of both WPBs, that were short and “stubby”, as well as the trans‐Golgi network (TGN). In addition, downmodulation of Sec22b expression resulted in the absence of HMW VWF multimers. Overexpression of a dominant‐negative Sec22b variant lacking its SNARE domain confirmed these phenotypes, revealing that correct localization and/or function of Sec22B is important for VWF trafficking and WPB formation.Summary/Conclusion:Our data suggest that Sec22b is a novel regulator of VWF trafficking and WPB biogenesis that modulates VWF multimerization and the elongated shape of WPBs by controlling ER/Golgi VWF influx.

Rab46 integrates Ca2+ and histamine signaling to regulate selective cargo release from Weibel-Palade bodies.

Endothelial cells selectively release cargo stored in Weibel-Palade bodies (WPBs) to regulate vascular function, but the underlying mechanisms are poorly understood. Here we show that histamine evokes the release of the proinflammatory ligand, P-selectin, while diverting WPBs carrying non-inflammatory cargo away from the plasma membrane to the microtubule organizing center. This differential trafficking is dependent on Rab46 (CRACR2A), a newly identified Ca2+-sensing GTPase, which localizes to a subset of P-selectin-negative WPBs. After acute stimulation of the H1 receptor, GTP-bound Rab46 evokes dynein-dependent retrograde transport of a subset of WPBs along microtubules. Upon continued histamine stimulation, Rab46 senses localized elevations of intracellular calcium and evokes dispersal of microtubule organizing center-clustered WPBs. These data demonstrate for the first time that a Rab GTPase, Rab46, integrates G protein and Ca2+ signals to couple on-demand histamine signals to selective WPB trafficking.

The von Willebrand factor D′D3 assembly and structural principles for factor VIII binding and concatemer biogenesis

AbstractD assemblies make up half of the von Willebrand factor (VWF), yet are of unknown structure. D1 and D2 in the prodomain and D′D3 in mature VWF at Golgi pH form helical VWF tubules in Weibel Palade bodies and template dimerization of D3 through disulfides to form ultralong VWF concatemers. D′D3 forms the binding site for factor VIII. The crystal structure of monomeric D′D3 with cysteine residues required for dimerization mutated to alanine was determined at an endoplasmic reticulum (ER)-like pH. The smaller C8-3, TIL3 (trypsin inhibitor-like 3), and E3 modules pack through specific interfaces as they wind around the larger, N-terminal, Ca2+-binding von Willebrand D domain (VWD) 3 module to form a wedge shape. D′ with its TIL′ and E′ modules projects away from D3. The 2 mutated cysteines implicated in D3 dimerization are buried, providing a mechanism for protecting them against premature disulfide linkage in the ER, where intrachain disulfide linkages are formed. D3 dimerization requires co-association with D1 and D2, Ca2+, and Golgi-like acidic pH. Associated structural rearrangements in the C8-3 and TIL3 modules are required to expose cysteine residues for disulfide linkage. Our structure provides insight into many von Willebrand disease mutations, including those that diminish factor VIII binding, which suggest that factor VIII binds not only to the N-terminal TIL′ domain of D′ distal from D3 but also extends across 1 side of D3. The organizing principle for the D3 assembly has implications for other D assemblies and the construction of higher-order, disulfide-linked assemblies in the Golgi in both VWF and mucins.

Novel Endothelial Cell Targeted Peptide Nanoformulation for Inhibiting von Willebrand Factor Secretion to Reduce Thrombotic Complications in Sepsis

RationaleMicrovascular thrombosis and disseminated intravascular coagulation (DIC) are primary determinants of ischemia, organ failure and mortality/morbidity in sepsis. Interestingly, elevated plasma level of von Willebrand factor (vWF), the glue to which platelets adhere to form a clot, is associated with reduced survival and poor outcome in septic patients. Previously, we demonstrated that endothelial cell (EC) Gα12 is required for soluble N‐ethylmaleimide‐sensitive‐fusion factor attachment protein α (αSNAP)‐dependent vWF secretion from Weibel Palade bodies. Here, we report that sterically stabilized simple micelles (SSM) containing cell‐permeable myristoylated αSNAP binding domain (Myr‐SBD) peptide based on the unique N‐terminal Gα12 sequence that binds to αSNAP, inhibits vWF secretion.MethodsStructural complex of αSNAP/SBD was determined by blind‐ and data‐driven docking using AutoDock and HADDOCK, respectively. After incorporating Myr‐SBD into SSM (Myr‐SBD‐SSM) by film hydration method, micelle size and shape were confirmed by dynamic light scattering and transmission electron microscopy (TEM), respectively. Intracellular delivery of Myr‐SBD‐SSM was evaluated by fluorescence microscopy and fluorometry in cultured ECs. Cecal ligation and puncture (CLP) in C57BL/6 mice (14–16 wks, 25 g) was used to induce severe inflammation, microvascular thrombosis and DIC, and blood was collected 6 h and 24 h post CLP. The effect of Myr‐SBD‐SSM on plasma vWF, platelet count and inflammatory cytokines was assessed following addition of 0, 2 or 4 μmol Myr‐SBD/kg given i.v. at the time of surgery. Bleeding was assessed by tail‐snip assay. Glomerular fibrin deposition was scored by IHC analysis at 24 h post CLP.ResultsThe αSNAP/SBD structure showed the potential interaction between Arg of SBD peptide and Glu139 of αSNAP by H‐bond. Spherical shape of Myr‐SBD‐SSM (14.1 ± 2.7 nm in diameter) was confirmed by negative staining TEM. Addition of Myr‐SBD‐SSM to ECs resulted in the intracellular accumulation of Myr‐SBD which blocked thrombin‐induced vWF secretion. Intravenous delivery of Myr‐SBD‐SSM to mice at the time of CLP blunted the increase in plasma vWF and associated microvascular thrombosis in the kidney glomerulus. Importantly, Myr‐SBD‐SSM treatment resulted in increased platelet and WBC counts 6 h after surgery and improved survival compared to untreated or scrambled peptide treated mice with severe vascular inflammation suggesting Myr‐SBD‐SSM reduced clotting, platelet consumption and systemic inflammation in intraperitoneal sepsis. Moreover, Myr‐SBD‐SSM treatment did not increase tail‐snip bleeding time suggesting it did not prevent hemostasis.ConclusionOur studies demonstrate that Myr‐SBD‐SSM nanoformulation reduces vWF secretion and attenuates the thrombotic complications of severe inflammation.Support or Funding InformationThis work was supported by MPI R01 HL125356 (RDM, XD), Michael Reese Research and Education Foundation (RDM) and American Heart Association (MB, LR).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Von Willebrand Factor Mediates Pneumococcal Aggregation and Adhesion in Blood Flow.

Streptococcus pneumoniae is a major cause of community acquired pneumonia and septicaemia in humans. These diseases are frequently associated with thromboembolic cardiovascular complications. Pneumococci induce the exocytosis of endothelial Weibel-Palade Bodies and thereby actively stimulate the release of von Willebrand factor (VWF), which is an essential glycoprotein of the vascular hemostasis. Both, the pneumococcus induced pulmonary inflammation and the thromboembolytic complications are characterized by a dysbalanced hemostasis including a marked increase in VWF plasma concentrations. Here, we describe for the first time VWF as a novel interaction partner of capsulated and non-encapsulated pneumococci. Moreover, cell culture infection analyses with primary endothelial cells characterized VWF as bridging molecule that mediates bacterial adherence to endothelial cells in a heparin-sensitive manner. Due to the mechanoresponsive changes of the VWF protein conformation and multimerization status, which occur in the blood stream, we used a microfluidic pump system to generate shear flow-induced multimeric VWF strings on endothelial cell surfaces and analyzed attachment of RFP-expressing pneumococci in flow. By applying immunofluorescence visualization and additional electron microscopy, we detected a frequent and enduring bacterial attachment to the VWF strings. Bacterial attachment to the endothelium was confirmed in vivo using a zebrafish infection model, which is described in many reports and acknowledged as suitable model to study hemostasis mechanisms and protein interactions of coagulation factors. Notably, we visualized the recruitment of zebrafish-derived VWF to the surface of pneumococci circulating in the blood stream and detected a VWF-dependent formation of bacterial aggregates within the vasculature of infected zebrafish larvae. Furthermore, we identified the surface-exposed bacterial enolase as pneumococcal VWF binding protein, which interacts with the VWF domain A1 and determined the binding kinetics by surface plasmon resonance. Subsequent epitope mapping using an enolase peptide array indicates that the peptide 181YGAEIFHALKKILKS195 might serve as a possible core sequence of the VWF interaction site. In conclusion, we describe a VWF-mediated mechanism for pneumococcal anchoring within the bloodstream via surface-displayed enolase, which promotes intravascular bacterial aggregation.

Histological and functional comparisons of four anatomical regions of porcine skin with human abdominal skin.

Because of the shortage of human skin for research purposes, porcine skin has been used as a model of human skin. The aim of this study was to identify the region of German Landrace pig skin that could be used as the best possible substitute for human abdominal skin. Porcine samples were collected from the ear, flank, back and caudal abdomen; human abdominal skin samples were excised during plastic surgery. Histological and ultrastructural assessments were carried out on the epidermis and dermis, with emphasis on the dermo-epidermal interface length, dermo-epidermal thickness ratio as well as densities of; hair follicles, arrector pili muscles, blood vessels and sweat glands. In the pig, the barrier function of the four anatomical regions was assessed. Results showed that both histologically and ultrastructurally, all four regions of porcine skin were similar to human skin. These include the shapes of keratinocytes, structure of cell contacts and presence of Weibel Palade bodies in endothelial cells. Other parameters such as the thickness of epidermis, the thickness of stratum basale, spinosum and granulosum and the number of cell layers in the stratum corneum were similar in human abdominal and in all four regions of porcine skin. However, there were also significant differences especially in the thickness of the stratum corneum, the dermo-epidermal interface length and the blood vessel density.

Defective AP-3-dependent VAMP8 trafficking impairs Weibel-Palade body exocytosis in Hermansky-Pudlak Syndrome type 2 blood outgrowth endothelial cells.

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.

Critical role of C5a in sickle cell disease.

Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia-reperfusion physiology is a key component of the inflammatory and vaso-occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro-inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion. We hypothesize that hypoxia-reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso-occlusion. At baseline and after H/R, sickle Townes-SS mice had increased C3 activation fragments and C5b-9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA-mice. Activated complement promoted vaso-occlusion (microvascular stasis) in SS-mice; infusion of zymosan-activated, but not heat-inactivated serum, induced substantial vaso-occlusion in the skin venules of SS-mice. Infusion of recombinant C5a induced stasis in SS, but not AA-mice that was blocked by anti-C5a receptor (C5aR) IgG. C5a-mediated stasis was accompanied by inflammatory responses in SS-mice including NF-κB activation and increased expression of TLR4 and adhesion molecules VCAM-1, ICAM-1, and E-selectin in the liver. Anti-C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up-regulated Weibel-Palade body P-selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS-mice, a blocking antibody to P-selectin inhibited C5a-induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R-induced stasis in SS-mice. These results suggest that inhibition of C5a may be beneficial in SCD.

Synaptotagmin 5 regulates Ca2+-dependent Weibel-Palade body exocytosis in human endothelial cells.

Elevations of intracellular free Ca2+ concentration ([Ca2+]i) are a potent trigger for Weibel-Palade body (WPB) exocytosis and secretion of von Willebrand factor (VWF) from endothelial cells; however, the identity of WPB-associated Ca2+-sensors involved in transducing acute increases in [Ca2+]i into granule exocytosis remains unknown. Here, we show that synaptotagmin 5 (SYT5) is expressed in human umbilical vein endothelial cells (HUVECs) and is recruited to WPBs to regulate Ca2+-driven WPB exocytosis. Western blot analysis of HUVECs identified SYT5 protein, and exogenously expressed SYT5-mEGFP localised almost exclusively to WPBs. shRNA-mediated knockdown of endogenous SYT5 (shSYT5) reduced the rate and extent of histamine-evoked WPB exocytosis and reduced secretion of the WPB cargo VWF-propeptide (VWFpp). The shSYT5-mediated reduction in histamine-evoked WPB exocytosis was prevented by expression of shRNA-resistant SYT5-mCherry. Overexpression of SYT5-EGFP increased the rate and extent of histamine-evoked WPB exocytosis, and increased secretion of VWFpp. Expression of a Ca2+-binding defective SYT5 mutant (SYT5-Asp197Ser-EGFP) mimicked depletion of endogenous SYT5. We identify SYT5 as a WPB-associated Ca2+ sensor regulating Ca2+-dependent secretion of stored mediators from vascular endothelial cells.

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.

Dengue Virus NS1 Exposure Affects von Willebrand Factor Profile and Platelet Adhesion Properties of Cultured Vascular Endothelial Cells.

Hematological abnormalities and altered vascular permeability are frequently encountered in Dengue virus infected patients, but the mechanisms that alter platelet-endothelium interactions remain incompletely understood. The DENV NS1 protein has been implicated in adverse disease outcomes. In the present study the role of NS1 protein in affecting the expression of vWF and platelet adhesion properties of endothelial cells was studied in vitro. The results suggest that vWF is down regulated in cultured endothelial cells 6 and 24h after exposure with increase in vWF levels in culture supernatants at corresponding time points. Ultrastructural studies showed distinct evidence of endothelial cell activation morphology and degranulation of Weibel-Palade bodies in NS1 exposed cells that also showed increased platelet activation physiology. The findings suggest that changes in vWF production and secretion may be induced in endothelial cells exposed to DENV NS1 protein; and play a role in bleeding complications of severe DENV disease.

Von Willebrand Factor in Angiogenesis and Angiodysplasia

von Willebrand factor (VWF) is best known for its key role in haemostasis, capturing platelets at sites of endothelial damage and acting as carrier for coagulation Factor VIII. The importance of VWF in haemostasis is illustrated by the fact that its deficiency and/or abnormality causes von Willebrand disease (VWD), the most frequent inherited bleeding disorder, whilst raised levels of VWF are associated with an increased risk of arterial thrombosis. VWF is synthesized in megakaryocytes and in endothelial cells from most, but not all, vascular districts. Besides the well characterized binding to Factor VIII and with platelet receptors, VWF can interact with a plethora of proteins, from extracellular matrix components to growth factors and even DNA, suggesting that VWF may influence multiple processes. Moreover, VWF is required for the formation of Weibel Palade Bodies (WPB), endothelial storage organelles which contain many vascular regulators. It is therefore likely that this large protein, critically located at sites of vascular injury, is able to influence several vascular functions. Indeed over the last two decades novel functions for VWF in the vasculature have been identified, including the ability to modulate blood vessel formation. Studies in a mouse models of severe VWF deficiency have shown constitutively enhanced vascular networks in selected tissues, and enhanced angiogenesis in Matrigel and in response to ischemia in the brain. Moreover, studies on circulating endothelial progenitors from patients with type 3 VWD and lack of VWF synthesis have shown enhanced in vitro angiogenesis. The ability of VWF to regulate angiogenesis has clinical implications for a subset of VWD patients with severe, intractable gastrointestinal (GI) bleeding due to vascular malformations, called angiodysplasia. These lesions, found in patients with congenital VWD and acquired von Willebrand syndrome (AVWS), can cause severe gastrointestinal bleeding, often unresponsive to conventional replacement therapy. Therefore, understanding the mechanisms through which VWF modulates blood vessel formation is likely to have direct implications for the treatment of these patients.In vitro and in vivo studies indicate that VWF can regulate angiogenesis through multiple pathways. Strong candidates for this role are VWF binding partners, such as integrin αvβ3, and components of Weibel Palade bodies (WPB), such as Angiopoietin-2 and Galectin-3, whose storage is regulated by VWF. Several of these pathways converge on the master regulator of angiogenesis, also essential for maintaining endothelial homeostasis, namely the vascular endothelial growth factor (VEGF) pathway. Multiple regulators may act in concert, their relevance differing in congenital VWD vs acquired AVWS. Interestingly, recent studies in mouse models suggest that the roles of VWF may be tissue-specific. If confirmed, this will have important implications for the translational and clinical implications of these findings for patient with VWD. In summary, the finding that VWF is able to regulate blood vessel formation has opened a new area of research for this incredibly interesting and versatile protein, one which has profound implications for the treatment of patients with VWD and AVWS. DisclosuresRandi:LFB: Other: Invited to one advisory board meeting 2018, Patents & Royalties, Research Funding, Speakers Bureau; Shire: Honoraria.

Actin dynamics during Ca2+-dependent exocytosis of endothelial Weibel-Palade bodies

Weibel-Palade bodies (WPBs) are specialized secretory organelles of endothelial cells that serve important functions in the response to inflammation and vascular injury. WPBs actively respond to different stimuli by regulated exocytosis leading to full or selective release of their contents. Cellular conditions and mechanisms that distinguish between these possibilities are only beginning to emerge. To address this we analyzed dynamic rearrangements of the actin cytoskeleton during histamine-stimulated, Ca2+-dependent WPB exocytosis. We show that most WPB fusion events are followed by a rapid release of von-Willebrand factor (VWF), the large WPB cargo, and that this occurs concomitant with a softening of the actin cortex by the recently described Ca2+-dependent actin reset (CaAR). However, a considerable fraction of WPB fusion events is characterized by a delayed release of VWF and observed after the CaAR reaction peak. These delayed VWF secretions are accompanied by an assembly of actin rings or coats around the WPB post-fusion structures and are also seen following direct elevation of intracellular Ca2+ by plasma membrane wounding. Actin ring/coat assembly at WPB post-fusion structures requires Rho GTPase activity and is significantly reduced upon expression of a dominant-active mutant of the formin INF2 that triggers a permanent CaAR peak-like sequestration of actin to the endoplasmic reticulum. These findings suggest that a rigid actin cortex correlates with a higher proportion of fused WPB which assemble actin rings/coats most likely required for efficient VWF expulsion and/or stabilization of a WPB post-fusion structure.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Pure ultra-fine carbon particles do not exert pro-coagulation and inflammatory effects on microvascular endothelial cells.

Pro-thrombotic and inflammatory changes play an important role in cardiovascular morbidity and mortality, resulting from short-term exposure to fine particulate air-pollution. Part of those effects has been attributed to the ultra-fine particles (UFPs) that pass through the lung and directly contact blood-exposed and circulating cells. Despite UFP-induced platelet activation, it is unclear whether the penetrated particles exert any direct effect on endothelial cells. While exposure levels are boosting as a result of world-wide increases in economic development and desertification, which create more air-polluted regions, as well as increase in demands for synthetic UFPs in medicine and various industries, further studies on the health effects of these particles are required. In this study, human pulmonary and cardiac microvascular endothelial cells (MECs) have been exposed to 0.1, 1, 10, and 100μg/ml suspensions of either a natural (carbon black) or a synthetic (multi-walled carbon nano-tubes) type of UFPs, in vitro. As a result, no changes in the levels of coagulation factor VIII, Von Willebrand factor, Interleukin 8, and P-selectin measured in the cells' supernatant were observed prior to and 6, 12, and 24h after exposure. In parallel, the spatio-temporal effect of UFPs on cardiac MECs was evaluated by Transmission Electron Microscopy. Despite phagocytic uptake of pure UFPs observed on cellular sections of the treated cells, Weibel-Palade bodies remained intact in shape and similar in number when compared with the untreated cells. Our work shows that carbon itself is a non-toxic carrier for endothelial cells.

Denisa Wagner.

Denisa Wagner.

Malaria pigment accelerates MTT - formazan exocytosis in human endothelial cells.

Haemozoin is a by-product of haemoglobin digestion by intraerythrocytic malaria parasites, which induces immunologic responses on different tissues, including endothelial cells. In the present paper, the incubation of human microvascular endothelial cells with haemozoin significantly inhibited MTT reduction, a measure of cytotoxicity, without increasing the release of cytoplasmic lactate dehydrogenase. Moreover, haemozoin did not induce apoptosis or cell cycle arrest nor decreased the number of live cells, suggesting that cells viability itself was not affected and that the inhibition of MTT reduction was only apparent and probably due to accelerated MTT-formazan exocytosis. After 30 min of MTT addition, a significant increase in the % of cells exocytosing MTT formazan crystals was observed in haemozoin-treated cells compared with control cells. Such an effect was partially reversed by the addition of genistein, an inhibitor of MTT-formazan exocytosis. The rapid release of CXCL-8, a preformed chemokine contained in Weibel-Palade bodies, confirmed that haemozoin induces a perturbation of the intracellular endothelial trafficking, including the exocytosis of MTT-formazan containing vesicles. The haem moiety of haemozoin is responsible for the observed effect. Moreover, this work underlines that MTT assay should not be used to measure cytotoxicity induced by haemozoin and other methods should be preferred.

The Involvement of Toll-like Receptor-2 in Arterial Thrombus Formation.

There is emerging evidence for the participation of toll-like receptor-2 (TLR2) expressed on platelets and endothelial cells in the setting of arterial thrombosis. In isolated human platelets, TLR2/1 activation was demonstrated to induce platelet activation, secretion, aggregation, adhesion to collagen coatings and the formation of platelet-leukocyte conjugates, whereas murine platelets were less sensitive to TLR2/1 stimulation. Also, endothelial cells can be activated by stimulation with TLR2 agonists, resulting in increased expression of adhesion molecules, synthesis of inflammatory mediators and Weibel-Palade body exocytosis. Endothelial TLR2 signalling promotes atherosclerotic lesion development in mouse atherosclerosis models. Experiments with germ-free mouse models demonstrated that the presence of commensal microbiota increased endothelial von Willebrand factor synthesis in the liver through TLR2. In the carotid artery ligation model, the elevated von Willebrand factor plasma levels enhanced platelet deposition to the injury site. Furthermore, in the hyperlipidemic ApoE-deficient mouse model, TLR2 deficiency was shown to protect from ferric chloride-induced carotid artery thrombosis. This review article provides an overview on TLR2 signalling in platelets and the vascular endothelium and summarizes how TLR2 signalling contributes to arterial thrombosis.

Tuning the endothelial response: differential release of exocytic cargos from Weibel‐Palade bodies

Essentials Endothelial activation initiates multiple processes, including hemostasis and inflammation.The molecules that contribute to these processes are co‐stored in secretory granules.How can the cells control release of granule content to allow differentiated responses?Selected agonists recruit an exocytosis‐linked actin ring to boost release of a subset of cargo. SummaryBackgroundEndothelial cells harbor specialized storage organelles, Weibel‐Palade bodies (WPBs). Exocytosis of WPB content into the vascular lumen initiates primary hemostasis, mediated by von Willebrand factor (VWF), and inflammation, mediated by several proteins including P‐selectin. During full fusion, secretion of this large hemostatic protein and smaller pro‐inflammatory proteins are thought to be inextricably linked.ObjectiveTo determine if secretagogue‐dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis.MethodsWe used VWF string analysis, leukocyte rolling assays, ELISA, spinning disk confocal microscopy, high‐throughput confocal microscopy and inhibitor and siRNA treatments to demonstrate the existence of cellular machinery that allows differential release of WPB cargo proteins.ResultsInhibition of the actomyosin ring differentially effects two processes regulated by WPB exocytosis; it perturbs VWF string formation but has no effect on leukocyte rolling. The efficiency of ring recruitment correlates with VWF release; the ratio of release of VWF to small cargoes decreases when ring recruitment is inhibited. The recruitment of the actin ring is time dependent (fusion events occurring directly after stimulation are less likely to initiate hemostasis than later events) and is activated by protein kinase C (PKC) isoforms.ConclusionsSecretagogues differentially recruit the actomyosin ring, thus demonstrating one mechanism by which the prothrombotic effect of endothelial activation can be modulated. This potentially limits thrombosis whilst permitting a normal inflammatory response. These results have implications for the assessment of WPB fusion, cargo‐content release and the treatment of patients with von Willebrand disease.