Endothelial Exocytosis Research Articles

RalB uncoupled exocyst mediates endothelial Weibel-Palade body exocytosis.

Ras-like (Ral) GTPases play essential regulatory roles in many cellular processes, including exocytosis. Cycling between GDP- and GTP-bound states, Ral GTPases function as molecular switches and regulate effectors, specifically the multi-subunit tethering complex exocyst. Here, we show that Ral isoform RalB controls regulated exocytosis of Weibel-Palade bodies (WPBs), the specialized endothelial secretory granules that store hemostatic protein von Willebrand factor. Remarkably, unlike typical small GTPase-effector interactions, RalB binds exocyst in its GDP-bound state in resting endothelium. Upon endothelial cell stimulation, exocyst is uncoupled from RalB-GTP resulting in WPB tethering and exocytosis. Furthermore, we report that PKC-dependent phosphorylation of the C-terminal hypervariable region (HVR) of RalB modulates its dynamic interaction with exocyst in endothelium. Exocyst preferentially interacts with phosphorylated RalB in resting endothelium. Dephosphorylation of RalB either by endothelial cell stimulation, or PKC inhibition, or expression of nonphosphorylatable mutant at a specific serine residue of RalB HVR, disengages exocyst and augments WPB exocytosis, resembling RalB exocyst-binding site mutant. In summary, it is the uncoupling of exocyst from RalB that mediates endothelial Weibel-Palade body exocytosis. Our data shows that Ral function may be more dynamically regulated by phosphorylation and may confer distinct functionality given high degree of homology and the shared set of effector protein between the two Ral isoforms.

Abstract 1063: G Protein 12 Alpha Subunit Covalent Binding To Alpha-snap And Its Potential Role In Von Willebrand Factor Secretion

Rationale: α-SNAP is part of a large 20S complex composed of 3 SNARE, 4 α-SNAP, and 6 N-ethylmaleimide sensitive factor proteins that together regulate endothelial cell Weibel Palade body fusion and exocytosis of von Willebrand factor (vWF). We previously showed that Gα12 is essential for basal and thrombin-induced vWF secretion, that GST-α-SNAP pulldown of Gα12 is dependent on N-terminal domain aa 10-15 in Gα12, and that a myristoylated 6 aa α-SNAP Binding Domain Gα12 peptide (Myr-SBD6) reduces vWF secretion, thrombo-inflammation, and lethality in septic mice. Here, using direct binding assays (Surface Plasmon Resonance (SPR), AlphaLISA, and mass spectrometry (MS)), we assessed the molecular underpinnings of SBD6 and modified higher affinity peptide (SBD15) binding directly to α-SNAP. Methods: GST-α-SNAP was incubated alone or with SBD6, SBD15-PEG-biotin, or SBD6 with C to S mutation and then evaluated by Maldi MS. SPR was performed for both SBD6 and SBD15 to assess binding affinity ( K D ) and kinetics ( k a and k d ). After demonstrating the covalent linkage of α-SNAP with SBD15-PEG-biotin, cysteine mapping was performed to identify the SBD binding site on α-SNAP. Free cysteines in SBD15-biotin conjugated α-SNAP were capped with N-ethylmaleimide, disulfide bonds were reduced with DTT, reduced Cys residues were capped with iodoacetamide (IAA), and tryptic peptide digests were analyzed by LC/MS/MS. Results: Maldi MS studies showed that SBD15-PEG-biotin exhibited covalent binding to GST-α-SNAP. Further, proteomic analysis of Cys mapped fragments identified a peptide containing C103, KADPQEAINC 103 LMR, that was modified by NEM in control samples and shifted in the presence of SBD15, indicating modification by IAA in α-SNAP - SBD15 conjugates. Further, the C103S α-SNAP mutant was unable to bind to SBD6, and C11S-SBD15-PEG-biotin was unable to bind to α-SNAP. Conclusion: We propose that amino-terminal Gα12 Cys11 covalently binds to α-SNAP Cys103 and that this plays a critical role in the mechanism regulating vWF secretion. The discovery of this disulfide binding mechanism between Gα12 and α-SNAP opens up new avenues for therapeutic intervention in thrombotic disorders associated with elevated plasma vWF.

Endothelial PTP1B Deletion Promotes VWF Exocytosis and Venous Thromboinflammation.

Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis. Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies. Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT2 PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V+ procoagulant endothelial CD62E+ (E-selectin) and neutrophil (Ly6G+) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082. Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment.

Microvascular activation and exocytosis after exposure to the serum from mismatched recipients by using donor microvascular cultures

BackgroundMicrovascular injury resulting from activation and exocytosis are early signs of tissue damage caused by allografting. However, humoral anti-graft reactions are not easily detectable in transplant biopsies. The aim of this study was to establish a bioassay to recapitulate this process in a prospective approach. MethodsThe study was executed by using our previously established protocol to isolate and freeze the donors' microvascular endothelial cells (MVEC) at the transplantation (34 living-related donors and 26 cadaver donors); and to collect sera from the recipients before the transplantation, one-, three- and six-months after transplantation. The activation and exocytosis of the MVEC were determined by incubating the donors' cultures with the recipients' sera. We determined if there was any endothelial activation by quantifying the releases of monocyte chemotactic protein-1 (MCP-1) and interleukin 8 (IL-8) in supernatants and the expressions of membrane intercellular adhesion molecule-1 (CD54) and intercellular adhesion molecule-1 (CD106) by flow cytometry. Endothelial exocytosis was determined by quantifying soluble E-selectin (CD62E) and cytoplasmic von Willebrand Factor (vWF) in supernatants. Endothelial activation or exocytosis was considered positive when the fold change (≧1.5) of post-transplantation to pre-transplantation was reached. We also monitored serum PRA and cytokines using Luminex multiple-plex and cytometric bead-based assay respectively. ResultsWe found 41.2% recipients (14 out of 34, ranging from 1.5 to 5.2 folds, p < 0.05) exhibited positive MVEC activation in the first month after transplantation as determined by IL-8 levels; 26.5% recipients (9 out of 34, ranging from 1.5 to 11.8 folds, p < 0.05) by MCP-1 levels. In the group of three months post-transplantation, 70.6% patients were positive (12 out of 17, ranging from 1.8 to 87.1 folds, p < 0.05) by IL-8 increased levels; 24% recipients (4 out of 17, ranging from 1.8 to 50.5 folds, p < 0.05) measured by MCP-1 levels. However, these changes disappeared six months after transplantation. Flow cytometric data showed that a time-dependent of CD54+ and CD106+ expressions existed over the course of six months. Most CD54+ and CD106+ cells were CD31- (platelet-endothelial cell adhesion molecule-1), though CD31+/CD106+ (37.5%, 3 out of 8) and CD31+/CD106+ (25%. 2 out of 8) were seen. When comparing donor MVEC activation to their recipient's proinflammatory cytokine levels or PRA status, we could not draw a conclusion regarding the connections between them. The sera collected from recipients at either one- or three-months after allografting did not significantly induce the release of either soluble CD62E or vWF (p > 0.05), indicating exocytosis was not significantly involved in the acute phase of allografting. ConclusionsThis bioassay enables us to detect the activation and exocytosis of donor MVEC elicited by respective sera from mismatched kidney recipients.

To Study the Correlation of Clinical Severity and Cytokine Storm in COVID-19 Pulmonary Embolism Patients by Using Computed Tomography Pulmonary Angiography (CTPA) Qanadli Clot Burden Scoring System.

Pulmonary embolism (PE) is a fatal form of venous thromboembolism (VTE), with an overall untreated mortality of up to 30%. Greater than 50% of patients with lower extremity proximal DVT have concurrent PE at presentation. VTE has been seen in up to one-third of patients with COVID-19 infections requiring intensive care unit (ICU) admission.The objective of this study is to determine the correlation between CT pulmonary angiography, pulmonary embolism clot burden, and the Qanadli scoring system with clinically severe COVID-19 pneumonia and cytokine storm. 153 COVID-19 hospitalized patients who underwent CT pulmonary angiography (CTPA) for likelyPE on pretest probability modified Wells criteriawere enrolled. COVID-19 pneumonia was classified as URTI (upper respiratory tract infection), mild, severe, and critical COVID pneumonia. For data analysis, we categorized into two groups: (1) the non-severe group included URTI and mild pneumonia, and (2) the severe group included severe and critical pneumonia. We used the Qanadli scoring system to assess the PE percentages of pulmonary vascular obstruction using CTPA. Results: 41.8% (64) of COVID-19 patients were diagnosed with pulmonary embolism (PE) on CTPA. The majority of 51.6% of pulmonary vascular occlusions using the Qanadli scoring system for pulmonary embolism were at segmental arterial levels. Out of 104 COVID-19 cytokine storm patients, 45 (43%)were associated with pulmonary embolism. Overall, a 25% (16) mortality rate wasobserved in COVID-19 patients with pulmonary embolism. The pathogenesis of hypercoagulability in COVID-19 may include direct endothelial cell invasion by the virus, microvascular inflammation, endothelial exocytosis, and endotheliitis. A meta-analysis of 71 studies to investigate the occurrence of PE on CTPA in COVID-19 patients found 48.6% in ICU settingsand 65.3% of patients have clots in the peripheral pulmonary vasculature. There is a significant correlation between pulmonary embolism and high clot burdenQanadli CTPA scores, as well as between the severity of COVID-19 pneumonia and mortality. The association between critically ill COVID-19 pneumonia and pulmonary embolism may result in higher mortality and a poor prognostic marker.

Aldosterone Increases Vascular Permeability in Rat Skin.

The aim of this study was to evaluate the effect of acute aldosterone (ALDO) administration on the vascular permeability of skin. ALDO was injected intradermally into rats, and vascular permeability was measured. Eplerenone (EPL), a selective mineralocorticoid receptor (MR) antagonist, was used. Skin biopsies were carried out for immunohistochemical (IHC) staining, and polymerase chain reactions were performed to analyze the expression of MR, 11β-hydroxysteroid dehydrogenase type 2, von Willebrand factor (vWF), vascular endothelial growth factor (VEGF), and zonula occludens 1. Our study showed the presence of MR in the rat skin vasculature for the first time. It was found that ALDO injection resulted in a more than 30% increase in vascular permeability and enhanced the endothelial exocytosis of vWF. The effect of ALDO diminished after EPL administration. An accumulation of vWF and a reduction in VEGF IHC staining were observed following chronic EPL administration. No effect of ALDO or EPL on the mRNA expression of the studied genes or skin structure was observed. The results suggest that ALDO increases vascular permeability in the skin via an MR-dependent mechanism. This effect of ALDO on skin microcirculation may have important therapeutic implications for diseases characterized by increased levels of ALDO and coexisting skin microangiopathy.

Von Willebrand disease and von Willebrand factor.

In the past several decades, knowledge of the underlying pathogenesis of von Willebrand disease (VWD) has increased tremendously, thanks in no small part to detailed genetic mapping of the von Willebrand Factor (VWF) gene and advances in genetic and bioinformatic technology. However, these advances do not always easily translate into improved management for patients with VWD and low-VWF levels. For example, the treatment of pregnant women with VWD both pre- and postpartum can be complicated. While knowledge of the VWF genotype at some amino acid positions can aid in knowledge of who may be at increased risk of thrombocytopenia or insufficient increase in VWF levels during pregnancy, in many cases, VWF levels and bleeding severity is highly heterogeneous, making monitoring recommended during pregnancy to optimize treatment strategies. VWF AND COVID-19: New challenges related to the consequences of dysregulation of hemostasis continue to be discovered. The ongoing COVID-19 pandemic has highlighted that VWF has additional biological roles in the regulation of inflammatory disorders and angiogenesis, disruption of which may contribute to COVID-19 induced vasculopathy. Increased endothelial cell activation and Weibel-Palade body exocytosis in severe COVID-19 lead to markedly increased plasma VWF levels. Coupled with impairment of normal ADAMTS13 multimer regulation, these data suggest a role for VWF in the pathogenesis underlying pulmonary microvascular angiopathy in severe COVID-19. With the increased affordability and availability of next-generation sequencing techniques, as well as a push towards a multi-omic approach and personalized medicine in human genetics, there is hope that translational research will improve VWD patient outcomes.

COVID-19 Sepsis: Pathogenesis and Endothelial Molecular Mechanisms Based on "Two-Path Unifying Theory" of Hemostasis and Endotheliopathy-Associated Vascular Microthrombotic Disease, and Proposed Therapeutic Approach with Antimicrothrombotic Therapy.

COVID-19 sepsis is characterized by acute respiratory distress syndrome (ARDS) as a consequence of pulmonary tropism of the virus and endothelial heterogeneity of the host. ARDS is a phenotype among patients with multiorgan dysfunction syndrome (MODS) due to disseminated vascular microthrombotic disease (VMTD). In response to the viral septicemia, the host activates the complement system which produces terminal complement complex C5b-9 to neutralize pathogen. C5b-9 causes pore formation on the membrane of host endothelial cells (ECs) if CD59 is underexpressed. Also, viral S protein attraction to endothelial ACE2 receptor damages ECs. Both affect ECs and provoke endotheliopathy. Disseminated endotheliopathy activates two molecular pathways: inflammatory and microthrombotic. The former releases inflammatory cytokines from ECs, which lead to inflammation. The latter initiates endothelial exocytosis of unusually large von Willebrand factor (ULVWF) multimers and FVIII from Weibel–Palade bodies. If ADAMTS13 is insufficient, ULVWF multimers activate intravascular hemostasis of ULVWF path. In activated ULVWF path, ULVWF multimers anchored to damaged endothelial cells recruit circulating platelets and trigger microthrombogenesis. This process produces “microthrombi strings” composed of platelet-ULVWF complexes, leading to endotheliopathy-associated VMTD (EA-VMTD). In COVID-19, microthrombosis initially affects the lungs per tropism causing ARDS, but EA-VMTD may orchestrate more complex clinical phenotypes, including thrombotic thrombocytopenic purpura (TTP)-like syndrome, hepatic coagulopathy, MODS and combined micro-macrothrombotic syndrome. In this pandemic, ARDS and pulmonary thromboembolism (PTE) have often coexisted. The analysis based on two hemostatic theories supports ARDS caused by activated ULVWF path is EA-VMTD and PTE caused by activated ULVWF and TF paths is macrothrombosis. The thrombotic disorder of COVID-19 sepsis is consistent with the notion that ARDS is virus-induced disseminated EA-VMTD and PTE is in-hospital vascular injury-related macrothrombosis which is not directly related to viral pathogenesis. The pathogenesis-based therapeutic approach is discussed for the treatment of EA-VMTD with antimicrothrombotic regimen and the potential need of anticoagulation therapy for coinciding macrothrombosis in comprehensive COVID-19 care.

Lysophosphatidylcholine in phospholipase A2-modified LDL triggers secretion of angiopoietin 2

Background and aimsSecretory phospholipase A2 (PLA2) hydrolyzes LDL phospholipids generating modified LDL particles (PLA2-LDL) with increased atherogenic properties. Exocytosis of Weibel–Palade bodies (WPB) releases angiopoietin 2 (Ang2) and externalizes P-selectin, which both play important roles in vascular inflammation.Here, we investigated the effects of PLA2-LDL on exocytosis of WPBs. MethodsHuman coronary artery endothelial cells (HCAECs) were stimulated with PLA2- LDL, and its uptake and effect on Ang2 release, leukocyte adhesion, and intracellular calcium levels were measured. The effects of PLA2-LDL on Ang2 release and WPB exocytosis were measured in and ex vivo in mice. ResultsExposure of HCAECs to PLA2-LDL triggered Ang2 secretion and promoted leukocyte-HCAEC interaction. Lysophosphatidylcholine was identified as a critical component of PLA2-LDL regulating the WPB exocytosis, which was mediated by cell-surface proteoglycans, phospholipase C, intracellular calcium, and cytoskeletal remodeling. PLA2-LDL also induced murine endothelial WPB exocytosis in blood vessels in and ex vivo, as evidenced by secretion of Ang2 in vivo, P-selectin translocation to plasma membrane in intact endothelial cells in thoracic artery and tracheal vessels, and reduced Ang2 staining in tracheal endothelial cells. Finally, in contrast to normal human coronary arteries, in which Ang2 was present only in the endothelial layer, at sites of advanced atherosclerotic lesions, Ang2 was detected also in the intima, media, and adventitia. ConclusionsOur studies reveal PLA2-LDL as a potent agonist of endothelial WPB exocytosis, resulting in increased secretion of Ang2 and translocation of P-selectin. The results provide mechanistic insight into PLA2-LDL-dependent promotion of vascular inflammation and atherosclerosis.

Estrogen activates endothelial exocytosis

Estrogen therapy is used to treat patients with post-menopausal symptoms, such as hot flashes and dyspareunia. Estrogen therapy also decreases the risk of fractures from osteoporosis in post-menopausal women. However, estrogen increases the risk of venous thromboembolic events, such as pulmonary embolism, but the pathways through which estrogen increase the risk of thromboembolism is unknown. Here, we show that estrogen elicits endothelial exocytosis, the key step in vascular thrombosis and inflammation. Exogenous 17β-estradiol (E2) stimulated endothelial exocytosis of Weibel-Palade bodies (WPBs), releasing von Willebrand factor (vWF) and interleukin-8 (IL-8). Conversely, the estrogen antagonist ICI-182,780 interfered with E2-induced endothelial exocytosis. The ERα agonist propyl pyrazole triol (PPT) but not the ERβ agonist diarylpropionitrile (DPN) induced vWF release, while ERα silencing counteracted vWF release by E2, suggesting that ERα mediates this effect. Exocytosis triggered by E2 occurred rapidly within 15 min and was not inhibited by either actinomycin D or cycloheximide. On the contrary, it was inhibited by the pre-treatment of U0126 or SB203580, an ERK or a p38 inhibitor, respectively, suggesting that E2-induced endothelial exocytosis is non-genomically mediated by the MAP kinase pathway. Finally, E2 treatment enhanced platelet adhesion to endothelial cells ex vivo, which was interfered with the pre-treatment of ICI-182,780 or U0126. Taken together, our data show that estrogen activates endothelial exocytosis non-genomically through the ERα-MAP kinase pathway. Our data suggest that adverse cardiovascular effects such as vascular inflammation and thrombosis should be considered in patients before menopausal hormone treatment.

Trafficking of JC virus-like particles across the blood-brain barrier.

Hollow viral vectors, such as John Cunningham virus-like particles (JC VLPs), provide a unique opportunity to deliver drug cargo into targeted cells and tissue. Current understanding of the entry of JC virus in brain cells has remained insufficient. In particular, interaction of JC VLPs with the blood–brain barrier (BBB) has not been analyzed in detail. Thus, JC VLPs were produced in this study for investigating the trafficking across the BBB. We performed a carotid artery injection procedure for mouse brain to qualitatively study JC VLPs' in vivo binding and distribution and used in vitro approaches to analyze their uptake and export kinetics in brain endothelial cells. Our results show that clathrin-dependent mechanisms contributed to the entry of VLPs into brain endothelial cells, and exocytosis or transcytosis of VLPs across the BBB was observed in vitro. VLPs were found to interact with sialic acid glycans in mouse brain endothelia. The ability of JC VLPs to cross the BBB can be useful in developing a delivery system for transport of genes and small molecule cargoes to the brain.

Induction of Ca2+-Dependent Exocytotic Processes by Laser Ablation of Endothelial Cells.

Ca2+ regulates a variety of cellular processes that are essential to maintain cell integrity and function. Different methods have been used to study these processes by increasing intracellular Ca2+ levels. Here, we describe a protocol to initiate Ca2+-dependent membrane-related events, using laser ablation by near-infrared irradiation. This creates a rupture in the plasma membrane that allows the extracellular Ca2+ to enter the cell and thereby induce a receptor-independent Ca2+ increase. We report laser ablation protocols to study two different Ca2+-induced processes in human endothelial cells-membrane resealing and exocytosis of secretory granulescalled Weibel-Palade bodies (WPBs). Thus, laser ablation represents a technique that permits the analysis of different Ca2+-regulated processes at high spatiotemporal resolution in a controlled manner.

Severe COVID-19 Is a Microvascular Disease.

Severe COVID-19 Is a Microvascular Disease.

Von Willebrand factor as a thrombotic and inflammatory mediator in critical illness.

The endothelial exocytosis of high-molecular-weight multimeric von Willebrand factor (vWF) may occur in critical illness states, including trauma and sepsis, leading to the sustained elevation and altered composition of plasma vWF. These critical illnesses involve the common process of sympathoadrenal activation and loss of the endothelial glycocalyx. As a prothrombotic and proinflammatory molecule that interacts with the endothelium, the alterations exhibited by vWF in critical illness have been implicated in the development and damaging effects of downstream pathologies, such as disseminated intravascular coagulation and systemic inflammatory response syndrome. Given the role of vWF in these pathologies, there has been a recent push to further understand how the molecule may be involved in the pathophysiology of related diseases, such as trauma-induced coagulopathy and acute renal injury, which are also known to develop secondarily to critical illness states. Elucidation of the role of vWF across the broader spectrum of generalized pathologies may provide a basis for the development of novel preventative and restorative measures, while also bolstering the scaffold of more widely used treatments, such as the administration of plasma-containing blood products.

Myosin IIa is the most important effector of zyxin in cAMP‐mediated endothelial exocytosis

Endothelial exocytosis, one of the first lines of protection against vascular injury, is critical to maintaining the homeostasis of the blood vessels. Weibel‐Palade bodies (WPBs)1, endothelium‐specific secretory granules, deliver hemostatic and inflammatory mediators including von Willebrand factor (VWF) and P‐selectin in response to a variety of agonists2. Recently, using live‐cell super‐resolution microscopy3, we visualize the dynamic interaction between fine actin filaments and WPBs and found previously unappreciated reorganization of pre‐existing actin filaments around WPBs before fusion4. In addition, we show that focal adhesion protein zyxin mediates the formation of actin frameworks on exocytic WPBs4. However, the detailed mechanism how zyxin regulates the reorganization of actin filaments is still unknown.In this report, we combined protein interactomics, genetic screening, high‐resolution imaging, and animal models, to show that the direct interaction between myosin IIa and zyxin is essential for cAMP‐mediated endothelial VWF secretion.Using affinity purification and co‐immunoprecipitation assay, we found that myosin IIa is the most abundant interacting protein of zyxin. Surprisingly, besides the zyxin‐related function on exocytosis, myosin IIa plays vital roles in the regulation of blood vessels homeostasis. Downregulation of myosin IIa by shRNA significantly suppresses both forskolin‐ and epinephrine‐induced VWF secretion. Using an inducible endothelial specific myosin IIa null mice model, we observed impaired epinephrine‐stimulated VWF release, prolonged bleeding time and thrombosis from those mice. Mechanistically, we found that in resting cells, the loss of myosin IIa, inhibition of myosin activity and blocking the phosphorylation of myosin IIa at S1943 decreased the peripheral localization of Rab27‐positive WPBs along stress fibers. Following stimulation with cAMP activating agents, myosin IIa interacted with zyxin and this required the phosphorylation of S1916 on myosin IIa mediated by CK2 to support the formation of functional actin framework, facilitating fusion and subsequent exocytosis.In summary, our work revealed that myosin IIa is the most important interacting protein of zyxin, which couples the maintenance of mature WPB in peripheral region to the promotion of VWF secretion in synergy with zyxin via spatiotemporally distinctive actin remodelling using different phosphorylation sites (As shown in Figure 1). Our findings implicate myosin IIa as an important regulator of blood homeostasis under pathophysiological conditions.Support or Funding Informationthe National Science Funds(No. 91339111, No. 81470298, and Project 31521062), the Major State Basic Research Development Program of China (No. 2012CB945103), and the National Science and Technology Support Project (grant no. 2014BAI02B01).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Zyxin regulates endothelial von Willebrand factor secretion by reorganizing actin filaments around exocytic granules

Endothelial exocytosis of Weibel–Palade body (WPB) is one of the first lines of defence against vascular injury. However, the mechanisms that control WPB exocytosis in the final stages (including the docking, priming and fusion of granules) are poorly understood. Here we show that the focal adhesion protein zyxin is crucial in this process. Zyxin downregulation inhibits the secretion of von Willebrand factor (VWF), the most abundant cargo in WPBs, from human primary endothelial cells (ECs) induced by cAMP agonists. Zyxin-deficient mice exhibit impaired epinephrine-stimulated VWF release, prolonged bleeding time and thrombosis, largely due to defective endothelial secretion of VWF. Using live-cell super-resolution microscopy, we visualize previously unappreciated reorganization of pre-existing actin filaments around WPBs before fusion, dependent on zyxin and an interaction with the actin crosslinker α-actinin. Our findings identify zyxin as a physiological regulator of endothelial exocytosis through reorganizing local actin network in the final stage of exocytosis.

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003.

BackgroundAntibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling.MethodsPrimary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003).ResultsTreatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement.ConclusionsDespite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR.

Endothelial exocytosis of angiopoietin-2 resulting from CCM3 deficiency contributes to cerebral cavernous malformation.

Cerebral cavernous malformations (CCMs) are vascular malformations that affect the central nervous system and result in cerebral hemorrhage, seizure and stroke. CCM arises from loss-of-function mutations in one of three genes: CCM1, CCM2 and CCM3 (PDCD10). CCM3 mutations in human often result in a more severe form of the disease, and CCM3 knockout mice show severe phenotypes with yet-to-be defined mechanisms. We have recently reported that CCM3 regulates UNC13 family-mediated exocytosis. Here we investigate endothelial cells (EC) exocytosis in CCM disease progression. We find that CCM3 suppresses UNC13B/VAMP3-dependent exocytosis of angiopoietin-2 (ANGPT2) in brain endothelial cells. CCM3 ablation in EC augments exocytosis and secretion of ANGPT2, correlating with destabilized EC junctions, enlarged lumen formation, and endothelial cell-pericyte dissociations. UNC13B deficiency that blunts ANGPT2 secretion from EC or an ANGPT2 neutralization antibody normalizes the defects caused by CCM3 deficiency. More importantly, ANGPT2 neutralization antibody treatment or UNC13B deficiency blunts the CCM lesion phenotypes, including disruption of EC junctions, vessel dilation and pericyte dissociation, in the brains and retinas caused by endothelial cell-specific CCM3 inactivation. Our study reveals that enhanced secretion of ANGPT2 in endothelial cells contributes to the progression of the CCM disease, providing a novel therapeutic approach to treat this devastating pathology.

Annexins A2 and A8 in endothelial cell exocytosis and the control of vascular homeostasis.

Blood vessel homeostasis is controlled by a variety of regulatory circuits that involve both the vessel-lining endothelial cells as well as the circulating blood cells and products thereof. One important feature is the control exerted by endothelial cells through regulated exocytosis of factors affecting blood coagulation and local inflammatory processes. These factors include two important adhesion proteins: the leukocyte receptor P-selectin and the pro-coagulant von Willebrand factor (VWF) that binds platelets and is involved in the formation of a platelet plug at sites of blood vessel injury. Failure to correctly produce and secrete P-selectin and VWF leads to pathologies such as von Willebrand disease, the most common inherited bleeding disorder. P-selectin and VWF are stored in unique secretory granules, the Weibel-Palade bodies (WPB), that undergo a complex maturation process and are acutely secreted following endothelial stimulation, e.g. in the course of inflammation or following blood vessel injury. Two annexins have been shown to be involved in different aspects of WPB biology: annexin A8 is required for proper WPB maturation and annexin A2 participates in late steps of WPB exocytosis. Thus, by affecting the stimulated release of P-selectin and VWF from endothelial cells, annexins fulfil important functions in the control of vascular homeostasis and could be considered as targets for influencing P-selectin- and VWF-dependent processes/pathologies.

A Novel Anti-Inflammatory Effect for High Density Lipoprotein.

High density lipoprotein has anti-inflammatory effects in addition to mediating reverse cholesterol transport. While many of the chronic anti-inflammatory effects of high density lipoprotein (HDL) are attributed to changes in cell adhesion molecules, little is known about acute signal transduction events elicited by HDL in endothelial cells. We now show that high density lipoprotein decreases endothelial cell exocytosis, the first step in leukocyte trafficking. ApoA-I, a major apolipoprotein of HDL, mediates inhibition of endothelial cell exocytosis by interacting with endothelial scavenger receptor-BI which triggers an intracellular protective signaling cascade involving protein kinase C (PKC). Other apolipoproteins within the HDL particle have only modest effects upon endothelial exocytosis. Using a human primary culture of endothelial cells and murine apo-AI knockout mice, we show that apo-AI prevents endothelial cell exocytosis which limits leukocyte recruitment. These data suggest that high density lipoprotein may inhibit diseases associated with vascular inflammation in part by blocking endothelial exocytosis.