430 results on '"Weibel-Palade bodies"'
Search Results
2. Is Endothelial Activation a Critical Event in Thrombotic Thrombocytopenic Purpura?
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Cauchois, Raphael, Muller, Romain, Lagarde, Marie, Dignat-George, Françoise, Tellier, Edwige, and Kaplanski, Gilles
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THROMBOTIC thrombocytopenic purpura , *ENDOTHELIAL cells , *COMPLEMENT activation , *CHROMATIN - Abstract
Thrombotic thrombocytopenic purpura (TTP) is a severe thrombotic microangiopathy. The current pathophysiologic paradigm suggests that the ADAMTS13 deficiency leads to Ultra Large-Von Willebrand Factor multimers accumulation with generation of disseminated microthrombi. Nevertheless, the role of endothelial cells in this pathology remains an issue. In this review, we discuss the various clinical, in vitro and in vivo experimental data that support the important role of the endothelium in this pathology, suggesting that ADAMTS13 deficiency may be a necessary but not sufficient condition to induce TTP. The "second hit" model suggests that in TTP, in addition to ADAMTS13 deficiency, endogenous or exogenous factors induce endothelial activation affecting mainly microvascular cells. This leads to Weibel–Palade bodies degranulation, resulting in UL-VWF accumulation in microcirculation. This endothelial activation seems to be worsened by various amplification loops, such as the complement system, nucleosomes and free heme. [ABSTRACT FROM AUTHOR]
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- 2023
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3. Biogenesis of lysosome-related organelles complex-2 is an evolutionarily ancient proto-coatomer complex.
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Thomason, Peter A., Corbyn, Ryan, Lilla, Sergio, Sumpton, David, Gilbey, Thomas, and Insall, Robert H.
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DICTYOSTELIUM , *LYSOSOMES , *EXOCYTOSIS , *ORGANELLES , *STRUCTURAL models - Abstract
Hermansky-Pudlak syndrome (HPS) is an inherited disorder of intracellular vesicle trafficking affecting the function of lysosome-related organelles (LROs). At least 11 genes underlie the disease, encoding four protein complexes, of which biogenesis of lysosome-related organelles complex-2 (BLOC-2) is the last whose molecular action is unknown. We find that the unicellular eukaryote Dictyostelium unexpectedly contains a complete BLOC-2, comprising orthologs of the mammalian subunits HPS3, -5, and -6, and a fourth subunit, an ortholog of the Drosophila LRO-biogenesis gene, Claret. Lysosomes from Dictyostelium BLOC-2 mutants fail to mature, similar to LROs from HPS patients, but for all endolysosomes rather than a specialized subset. They also strongly resemble lysosomes from WASH mutants. Dictyostelium BLOC-2 localizes to the same compartments as WASH, and in BLOC-2 mutants, WASH is inefficiently recruited, accounting for their impaired lysosomal maturation. BLOC-2 is recruited to endolysosomes via its HPS3 subunit. Structural modeling suggests that all four subunits are proto-coatomer proteins, with important implications for BLOC-2's molecular function. The discovery of Dictyostelium BLOC-2 permits identification of orthologs throughout eukaryotes. BLOC-2 and lysosome-related organelles, therefore, pre-date the evolution of Metazoa and have broader and more conserved functions than previously thought. [Display omitted] • Dictyostelium BLOC-2 comprises orthologs of the mammalian subunits, plus Claret • BLOC-2 mutants have disrupted endolysosome maturation, leading to delayed exocytosis • BLOC-2 functions at the same compartment as WASH, but genetically upstream • BLOC-2 subunit genes are found in diverse eukaryotes but may have been widely lost Thomason et al. discover that biogenesis of lysosome-related organelles complex-2 (BLOC-2) is an ancient proto-coatomer complex widely conserved throughout eukaryotes. In the unicellular Dictyostelium , it functions in a central endolysosomal vesicle maturation pathway, perhaps reflective of its role in the last eukaryotic common ancestor. [ABSTRACT FROM AUTHOR]
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- 2024
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4. Endothelial colony-forming cells in the spotlight: insights into the pathophysiology of von Willebrand disease and rare bleeding disorders.
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Laan SNJ, Lenderink BG, Eikenboom JCJ, and Bierings R
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Endothelial cells deliver a vital contribution to the maintenance of hemostasis by constituting an anatomical as well as functional barrier between the blood and the rest of the body. Apart from the physical barrier function, endothelial cells maintain the hemostatic equilibrium by their pro- and anticoagulant functions. An important part of their procoagulant contribution is the production of von Willebrand factor (VWF), which is a carrier protein for coagulation factor VIII and facilitates the formation of a platelet plug. Thus, VWF is indispensable for both primary and secondary hemostasis, which is exemplified by the bleeding disorder von Willebrand disease that results from qualitative or quantitative deficiencies in VWF. A cellular model that was found to accurately reflect the endothelium and its secretory organelles are endothelial colony-forming cells, which can be readily isolated from peripheral blood and constitute a robust ex vivo model to investigate the donor's endothelial cell function. This review summarizes some of the valuable insights on biology of VWF and pathogenic mechanisms of von Willebrand disease that have been made possible using studies with endothelial colony-forming cells derived from patients with bleeding disorders., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
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- 2024
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5. Tip-end fusion of a rod-shaped secretory organelle.
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Naß, Johannes, Koerdt, Sophia N., Biesemann, Anja, Chehab, Tarek, Yasuda, Takao, Fukuda, Mitsunori, Martín-Belmonte, Fernando, and Gerke, Volker
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Weibel–Palade bodies (WPB) are elongated, rod-like secretory organelles unique to endothelial cells that store the pro-coagulant von-Willebrand factor (VWF) and undergo regulated exocytosis upon stimulation with Ca2+- or cAMP-raising agonists. We show here that WPB preferentially initiate fusion with the plasma membrane at their tips and identify synaptotagmin-like protein 2-a (Slp2-a) as a positive regulator of VWF secretion most likely mediating this topological selectivity. Following secretagogue stimulation, Slp2-a accumulates at one WPB tip before fusion occurs at this site. Depletion of Slp2-a reduces Ca2+-dependent secretion of highly multimeric VWF and interferes with the formation of actin rings at WPB–plasma membrane fusion sites that support the expulsion of the VWF multimers and most likely require a tip-end fusion topology. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] binding via the C2A domain of Slp2-a is required for accumulation of Slp2-a at the tip ends of fusing WPB, suggesting that Slp2-a mediates polar exocytosis by initiating contacts between WPB tips and plasma membrane PI(4,5)P2. [ABSTRACT FROM AUTHOR]
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- 2022
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6. A novel Munc13-4/S100A10/annexin A2 complex promotes Weibel-Palade body exocytosis in endothelial cells.
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Chehab, Tarek, Santos, Nina, Holthenrich, Anna, Koerdt, Sophia, Disse, Jennifer, Schuberth, Christian, Nazmi, Ali, Neeft, Maaike, Koch, Henriette, Wojcik, Sonja, Martin, Thomas, van der Sluijs, Peter, Brose, Nils, Gerke, Volker, and Man, Kwun Nok Mimi
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Annexin A2 ,Cell Membrane ,Cells ,Cultured ,Endothelial Cells ,Exocytosis ,Histamine ,Human Umbilical Vein Endothelial Cells ,Humans ,Membrane Proteins ,Protein Binding ,Protein Transport ,S100 Proteins ,Weibel-Palade Bodies ,von Willebrand Factor - Abstract
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.
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- 2017
7. A Homozygous Deep Intronic Variant Causes Von Willebrand Factor Deficiency and Lack of Endothelial-Specific Secretory Organelles, Weibel–Palade Bodies.
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Yadegari, Hamideh, Jamil, Muhammad Ahmer, Marquardt, Natascha, and Oldenburg, Johannes
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VON Willebrand factor , *VON Willebrand disease , *RNA analysis , *NUCLEOTIDE sequencing , *ORGANELLES , *ENDOTHELIAL cells - Abstract
A type 3 von Willebrand disease (VWD) index patient (IP) remains mutation-negative after completion of the conventional diagnostic analysis, including multiplex ligation-dependent probe amplification and sequencing of the promoter, exons, and flanking intronic regions of the VWF gene (VWF). In this study, we intended to elucidate causative mutation through next-generation sequencing (NGS) of the whole VWF (including complete intronic region), mRNA analysis, and study of the patient-derived endothelial colony-forming cells (ECFCs). The NGS revealed a variant in the intronic region of VWF (997 + 118 T > G in intron 8), for the first time. The bioinformatics assessments (e.g., SpliceAl) predicted this variant creates a new donor splice site (ss), which could outcompete the consensus 5′ donor ss at exon/intron 8. This would lead to an aberrant mRNA that contains a premature stop codon, targeting it to nonsense-mediated mRNA decay. The subsequent quantitative real-time PCR confirmed the virtual absence of VWF mRNA in IP ECFCs. Additionally, the IP ECFCs demonstrated a considerable reduction in VWF secretion (~6% of healthy donors), and they were devoid of endothelial-specific secretory organelles, Weibel–Palade bodies. Our findings underline the potential of NGS in conjunction with RNA analysis and patient-derived cell studies for genetic diagnosis of mutation-negative type 3 VWD patients. [ABSTRACT FROM AUTHOR]
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- 2022
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8. Spire1 and Myosin Vc promote Ca2+-evoked externalization of von Willebrand factor in endothelial cells.
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Holthenrich, Anna, Terglane, Julian, Naß, Johannes, Mietkowska, Magdalena, Kerkhoff, Eugen, and Gerke, Volker
- Abstract
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. [ABSTRACT FROM AUTHOR]
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- 2022
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9. Lowering the increased intracellular pH of human‐induced pluripotent stem cell‐derived endothelial cells induces formation of mature Weibel‐Palade bodies
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Gesa L. Tiemeier, Rozemarijn deKoning, Gangqi Wang, Sarantos Kostidis, Rosalie G. J. Rietjens, Wendy M. P. J. Sol, Sébastien J. Dumas, Martin Giera, Cathelijne W. van denBerg, Jeroen C. J. Eikenboom, Bernard M. van denBerg, Peter Carmeliet, and Ton J. Rabelink
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cell therapy ,glycolysis ,induced pluripotent stem cell‐derived endothelial cells ,von Willebrand factor ,Weibel‐Palade bodies ,Medicine (General) ,R5-920 ,Cytology ,QH573-671 - Abstract
Abstract Differentiation of human‐induced pluripotent stem cells (hiPSCs) into vascular endothelium is of great importance to tissue engineering, disease modeling, and use in regenerative medicine. Although differentiation of hiPSCs into endothelial‐like cells (hiPSC‐derived endothelial cells [hiPSC‐ECs]) has been demonstrated before, controversy exists as to what extent these cells faithfully reflect mature endothelium. To address this issue, we investigate hiPSC‐ECs maturation by their ability to express von Willebrand factor (VWF) and formation of Weibel‐Palade bodies (WPBs). Using multiple hiPSCs lines, hiPSC‐ECs failed to form proper VWF and WPBs, essential for angiogenesis, primary and secondary homeostasis. Lowering the increased intracellular pH (pHi) of hiPSC‐ECs with acetic acid did result in the formation of elongated WPBs. Nuclear magnetic resonance data showed that the higher pHi in hiPSC‐ECs occurred in association with decreased intracellular lactate concentrations. This was explained by decreased glycolytic flux toward pyruvate and lactate in hiPSC‐ECs. In addition, decreased expression of monocarboxylate transporter member 1, a member of the solute carrier family (SLC16A1), which regulates lactate and H+ uptake, contributed to the high pHi of hiPSC‐EC. Mechanistically, pro‐VWF dimers require the lower pH environment of the trans‐Golgi network for maturation and tubulation. These data show that while hiPSC‐ECs may share many features with mature EC, they are characterized by metabolic immaturity hampering proper EC function.
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- 2020
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10. Shrinking Weibel‐Palade bodies prevents high platelet recruitment in assays using thrombotic thrombocytopenic purpura plasma
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Francesca Patella, Chiara Vendramin, Oscar Charles, Marie A. Scully, and Daniel F. Cutler
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blood platelets ,fluvastatin ,human ,thrombotic thrombocytopenic purpura ,von Willebrand factor ,Weibel‐Palade bodies ,Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Abstract Background Thrombotic thrombocytopenic purpura (TTP), caused by a genetic or autoimmune‐driven lack of ADAMTS‐13 activity, leads to high levels of the ultra‐large von Willebrand factor (VWF) multimers produced by endothelial cells, causing excess platelet recruitment into forming thrombi, often with mortal consequences. Treatments include plasma infusion or replacement to restore ADAMTS‐13 activity, or prevention of platelet recruitment to VWF. Objectives We tested a different approach, exploiting the unique cell biology of the endothelium. Upon activation, the VWF released by exocytosis of Weibel‐Palade bodies (WPBs), transiently anchored to the cell surface, unfurls as strings into flowing plasma, recruiting platelets. Using plasma from patients with TTP increases platelet recruitment to the surface of cultured endothelial cells under flow. WPBs are uniquely plastic, and shortening WPBs dramatically reduces VWF string lengths and the recruitment of platelets. We wished to test whether the TTP plasma‐driven increase in platelet recruitment would be countered by reducing formation of the longest WPBs that release longer strings. Methods Endothelial cells grown in flow chambers were treated with fluvastatin, one of 37 drugs shown to shorten WPBs, then activated under flow in the presence of platelets and plasma of either controls or patients with TTP. Result We found that the dramatic increase in platelet recruitment caused by TTP plasma is entirely countered by treatment with fluvastatin, shortening the WPBs. Conclusions This potential approach of ameliorating the endothelial contribution to thrombotic risk by intervening far upstream of hemostasis might prove a useful adjunct to more conventional and direct therapies.
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- 2021
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11. Human Umbilical Vein Endothelial Cells as a Versatile Cellular Model System in Diverse Experimental Paradigms: An Ultrastructural Perspective.
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Duranova H, Kuzelova L, Borotova P, Simora V, and Fialkova V
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- Humans, Organelles ultrastructure, Human Umbilical Vein Endothelial Cells, Microscopy, Electron, Transmission
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Human umbilical vein endothelial cells (HUVECs) are primary cells isolated from the vein of an umbilical cord, extensively used in cardiovascular studies and medical research. These cells, retaining the characteristics of endothelial cells in vivo, serve as a valuable cellular model system for understanding vascular biology, endothelial dysfunction, pathophysiology of diseases such as atherosclerosis, and responses to different drugs or treatments. Transmission electron microscopy (TEM) has been a cornerstone in revealing the detailed architecture of multiple cellular model systems including HUVECs, allowing researchers to visualize subcellular organelles, membrane structures, and cytoskeletal elements. Among them, the endoplasmic reticulum, Golgi apparatus, mitochondria, and nucleus can be meticulously examined to recognize alterations indicative of cellular responses to various stimuli. Importantly, Weibel-Palade bodies are characteristic secretory organelles found in HUVECs, which can be easily distinguished in the TEM. These distinctive structures also dynamically react to different factors through regulated exocytosis, resulting in complete or selective release of their contents. This detailed review summarizes the ultrastructural features of HUVECs and highlights the utility of TEM as a pivotal tool for analyzing HUVECs in diverse research frameworks, contributing valuable insights into the comprehension of HUVEC behavior and enriching our knowledge into the complexity of vascular biology., Competing Interests: Conflict of Interest The authors declare that they have no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Microscopy Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)
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- 2024
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12. Transcriptional and functional profiling identifies inflammation and endothelial-to-mesenchymal transition as potential drivers for phenotypic heterogeneity within a cohort of endothelial colony forming cells.
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Laan SNJ, de Boer S, Dirven RJ, van Moort I, Kuipers TB, Mei H, Bierings R, and Eikenboom J
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- Humans, Cells, Cultured, Endothelial Cells metabolism, Endothelial Cells pathology, Epithelial-Mesenchymal Transition, Cell Proliferation, Endothelial Progenitor Cells metabolism, Endothelial Progenitor Cells pathology, Male, Female, Middle Aged, Adult, Transcription, Genetic, Phenotype, Gene Expression Profiling, Cell Movement, Inflammation genetics, Transcriptome
- Abstract
Background: Endothelial colony-forming cells (ECFCs) derived from patients can be used to investigate pathogenic mechanisms of vascular diseases like von Willebrand disease. Considerable phenotypic heterogeneity has been observed between ECFC clones derived from healthy donors. This heterogeneity needs to be well understood in order to use ECFCs as endothelial models for disease., Objectives: Therefore, we aimed to determine phenotypic and gene expression differences between control ECFCs., Methods: A total of 34 ECFC clones derived from 16 healthy controls were analyzed. The transcriptome of a selection of ECFC clones (n = 15) was analyzed by bulk RNA sequencing and gene set enrichment analysis. Gene expression was measured in all ECFC clones by quantitative polymerase chain reaction. Phenotypic profiling was performed and migration speed of the ECFCs was measured using confocal microscopy, followed by automated quantification of cell morphometrics and migration speed., Results: Through hierarchical clustering of RNA expression profiles, we could distinguish 2 major clusters within the ECFC cohort. Major differences were associated with proliferation and migration in cluster 1 and inflammation and endothelial-to-mesenchymal transition in cluster 2. Phenotypic profiling showed significantly more and smaller ECFCs in cluster 1, which contained more and longer Weibel-Palade bodies. Migration speed in cluster 1 was also significantly higher., Conclusion: We observed a range of different RNA expression patterns between ECFC clones, mostly associated with inflammation and clear differences in Weibel-Palade body count and structure. We developed a quantitative polymerase chain reaction panel that can be used for the characterization of ECFC clones, which is essential for the correct analysis of pathogenic mechanisms in vascular disorders., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
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- 2024
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13. A new look at an old body: molecular determinants of Weibel-Palade body composition and von Willebrand factor exocytosis.
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Hordijk S, Carter T, and Bierings R
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- Humans, Animals, Proteomics methods, Hemostasis, Weibel-Palade Bodies metabolism, Exocytosis, von Willebrand Factor metabolism, Endothelial Cells metabolism
- Abstract
Endothelial cells, forming a monolayer along blood vessels, intricately regulate vascular hemostasis, inflammatory responses, and angiogenesis. A key determinant of these functions is the controlled secretion of Weibel-Palade bodies (WPBs), which are specialized endothelial storage organelles housing a presynthesized pool of the hemostatic protein von Willebrand factor and various other hemostatic, inflammatory, angiogenic, and vasoactive mediators. This review delves into recent mechanistic insights into WPB biology, including the biogenesis that results in their unique morphology, the acquisition of intraluminal vesicles and other cargo, and the contribution of proton pumps to organelle acidification. Additionally, in light of a number of proteomic approaches to unravel the regulatory networks that control WPB formation and secretion, we provide a comprehensive overview of the WPB exocytotic machinery, including their molecular and cellular mechanisms., Competing Interests: Declaration of competing interests The authors state that they have no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
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- 2024
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14. It’s So 'Vonderful'
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Friedman, Mark T., West, Kamille A., Bizargity, Peyman, Annen, Kyle, Jhang, Jeffrey S., Friedman, Mark T., West, Kamille A., Bizargity, Peyman, Annen, Kyle, and Jhang, Jeffrey S.
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- 2018
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15. The Golgi Is a Measuring Cup
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Marshall, Wallace F
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Biochemistry and Cell Biology ,Biological Sciences ,Underpinning research ,1.1 Normal biological development and functioning ,Generic health relevance ,Human Umbilical Vein Endothelial Cells ,Humans ,Weibel-Palade Bodies ,trans-Golgi Network ,von Willebrand Factor ,Medical and Health Sciences ,Developmental Biology ,Biochemistry and cell biology - Abstract
The relation of organelle size to cellular function is a basic question in cell biology about which almost nothing is known. Reporting in this issue of Developmental Cell, Ferraro et al. (2014) show that the size and topology of the Golgi apparatus determines the size and functionality of a medically important secretory granule.
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- 2014
16. A Homozygous Deep Intronic Variant Causes Von Willebrand Factor Deficiency and Lack of Endothelial-Specific Secretory Organelles, Weibel–Palade Bodies
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Hamideh Yadegari, Muhammad Ahmer Jamil, Natascha Marquardt, and Johannes Oldenburg
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von Willebrand factor ,von Willebrand disease ,deep intronic mutation ,next-generation sequencing ,Weibel–Palade bodies ,angiopoietin-2 ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
A type 3 von Willebrand disease (VWD) index patient (IP) remains mutation-negative after completion of the conventional diagnostic analysis, including multiplex ligation-dependent probe amplification and sequencing of the promoter, exons, and flanking intronic regions of the VWF gene (VWF). In this study, we intended to elucidate causative mutation through next-generation sequencing (NGS) of the whole VWF (including complete intronic region), mRNA analysis, and study of the patient-derived endothelial colony-forming cells (ECFCs). The NGS revealed a variant in the intronic region of VWF (997 + 118 T > G in intron 8), for the first time. The bioinformatics assessments (e.g., SpliceAl) predicted this variant creates a new donor splice site (ss), which could outcompete the consensus 5′ donor ss at exon/intron 8. This would lead to an aberrant mRNA that contains a premature stop codon, targeting it to nonsense-mediated mRNA decay. The subsequent quantitative real-time PCR confirmed the virtual absence of VWF mRNA in IP ECFCs. Additionally, the IP ECFCs demonstrated a considerable reduction in VWF secretion (~6% of healthy donors), and they were devoid of endothelial-specific secretory organelles, Weibel–Palade bodies. Our findings underline the potential of NGS in conjunction with RNA analysis and patient-derived cell studies for genetic diagnosis of mutation-negative type 3 VWD patients.
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- 2022
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17. Mutations in Neurobeachin-like 2 do not impact Weibel-Palade body biogenesis and von Willebrand factor secretion in gray platelet syndrome Endothelial Colony Forming Cells
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Marije Kat, Iris van Moort, Petra E. Bürgisser, Taco W. Kuijpers, Menno Hofman, Marie Favier, Rémi Favier, Coert Margadant, Jan Voorberg, Ruben Bierings, Landsteiner Laboratory, Paediatric Infectious Diseases / Rheumatology / Immunology, AII - Infectious diseases, AII - Inflammatory diseases, AR&D - Amsterdam Reproduction & Development, Experimental Vascular Medicine, ACS - Microcirculation, Hematology, Pediatrics, and Medical oncology laboratory
- Subjects
gray platelet syndrome ,NBEAL2 protein ,Weibel-Palade bodies ,Hematology ,von Willebrand factor ,endothelial cells ,SEC22B - Abstract
Background: Patients with gray platelet syndrome (GPS) and Neurobeachin-like 2 (NBEAL2) deficiency produce platelets lacking alpha-granules (AGs) and present with lifelong bleeding symptoms. AGs are lysosome-related organelles and store the hemostatic protein von Willebrand factor (VWF) and the transmembrane protein P-selectin. Weibel-Palade bodies (WPBs) are lysosome-related organelles of endothelial cells and also store VWF and P-selectin. In megakaryocytes, NBEAL2 links P-selectin on AGs to the SNARE protein SEC22B on the endoplasmic reticulum, thereby preventing premature release of cargo from AG precursors. In endothelial cells, SEC22B drives VWF trafficking from the endoplasmic reticulum to Golgi and promotes the formation of elongated WPBs, but it is unclear whether this requires NBEAL2. Objectives: To investigate a potential role for NBEAL2 in WPB biogenesis and VWF secretion using NBEAL2-deficient endothelial cells. Methods: The interaction of SEC22B with NBEAL2 in endothelial cells was investigated by interatomic mass spectrometry and pull-down analysis. Endothelial colony forming cells were isolated from healthy controls and 3 unrelated patients with GPS and mutations in NBEAL2. Results: We showed that SEC22B binds to NBEAL2 in ECs. Endothelial colony forming cells derived from a patient with GPS are deficient in NBEAL2 but reveal normal formation and maturation of WPBs and normal WPB cargo recruitment. Neither basal nor histamine-induced VWF secretion is altered in the absence of NBEAL2. Conclusions: Although NBEAL2 deficiency causes the absence of AGs in patients with GPS, it does not impact WPB functionality in ECs. Our data highlight the differences in the regulatory mechanisms between these 2 hemostatic storage compartments.
- Published
- 2023
18. Pure ultra-fine carbon particles do not exert pro-coagulation and inflammatory effects on microvascular endothelial cells.
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Dinmohammadi, Hossein, Pirdel, Zahra, Salarilak, Laleh, Hoylaerts, Marc, Nejatbakhsh, Reza, Biglari, Alireza, Jacquemin, Marc, and Shahani, Tina
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AIR pollution ,PARTICULATE matter ,ENDOTHELIAL cells ,BLOOD platelets ,PHAGOCYTOSIS - Abstract
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 100 μg/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 24 h 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. [ABSTRACT FROM AUTHOR]
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- 2019
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19. Single-cell transcriptional analysis of human endothelial colony-forming cells from patients with low VWF levels
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Christopher J. Ng, Alice Liu, Sujatha Venkataraman, Katrina J. Ashworth, Christopher D. Baker, Rebecca O’Rourke, Rajeev Vibhakar, Kenneth L. Jones, and Jorge Di Paola
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congenital, hereditary, and neonatal diseases and abnormalities ,Weibel-Palade Bodies ,Immunology ,Cell Biology ,Hematology ,Biochemistry ,von Willebrand Diseases ,hemic and lymphatic diseases ,von Willebrand Factor ,Human Umbilical Vein Endothelial Cells ,cardiovascular system ,Humans ,Blood Commentary ,RNA, Small Interfering ,Single-Cell Analysis ,circulatory and respiratory physiology - Abstract
von Willebrand factor (VWF) plays a key role in normal hemostasis, and deficiencies of VWF lead to clinically significant bleeding. We sought to identify novel modifiers of VWF levels in endothelial colony-forming cells (ECFCs) using single-cell RNA sequencing (scRNA-seq). ECFCs were isolated from patients with low VWF levels (plasma VWF antigen levels between 30 and 50 IU/dL) and from healthy controls. Human umbilical vein endothelial cells were used as an additional control cell line. Cells were characterized for their Weibel Palade body (WPB) content and VWF release. scRNA-seq of all cell lines was performed to evaluate for gene expression heterogeneity and for candidate modifiers of VWF regulation. Candidate modifiers identified by scRNA-seq were further characterized with small-interfering RNA (siRNA) experiments to evaluate for effects on VWF. We observed that ECFCs derived from patients with low VWF demonstrated alterations in baseline WPB metrics and exhibit impaired VWF release. scRNA-seq analyses of these endothelial cells revealed overall decreased VWF transcription, mosaicism of VWF expression, and genes that are differentially expressed in low VWF ECFCs and control endothelial cells (control ECs). An siRNA screen of potential VWF modifiers provided further evidence of regulatory candidates, and 1 such candidate, FLI1, alters the transcriptional activity of VWF. In conclusion, ECFCs from individuals with low VWF demonstrate alterations in their baseline VWF packaging and release compared with control ECs. scRNA-seq revealed alterations in VWF transcription, and siRNA screening identified multiple candidate regulators of VWF.
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- 2022
20. Dispatch and delivery at the ER–Golgi interface: how endothelial cells tune their hemostatic response
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Marije Kat, Coert Margadant, Jan Voorberg, Ruben Bierings, Graduate School, ACS - Microcirculation, Other Research, AII - Inflammatory diseases, Experimental Vascular Medicine, Landsteiner Laboratory, Medical oncology laboratory, and Cancer Center Amsterdam
- Subjects
congenital, hereditary, and neonatal diseases and abnormalities ,Weibel-Palade Bodies ,Von Willebrand disease ,Endothelial Cells ,Von Willebrand factor ,Cell Biology ,Biochemistry ,Hemostatics ,von Willebrand Diseases ,endoplasmic reticulum ,SNARE ,hemic and lymphatic diseases ,cardiovascular system ,Golgi ,endothelial cell ,Humans ,Weibel-Palade body ,GBF1 ,Molecular Biology ,STX5 ,circulatory and respiratory physiology ,SEC22B - Abstract
Von Willebrand factor (VWF) is a glycoprotein that is secreted into the circulation and controls bleeding by promoting adhesion and aggregation of blood platelets at sites of vascular injury. Substantial inter-individual variation in VWF plasma levels exists among the healthy population. Prior to secretion, VWF polymers are assembled and condensed into helical tubules, which are packaged into Weibel-Palade bodies (WPBs), a highly specialized post-Golgi storage compartment in vascular endothelial cells. In the inherited bleeding disorder Von Willebrand disease (VWD), mutations in the VWF gene can cause qualitative or quantitative defects, limiting protein function, secretion, or plasma survival. However, pathogenic VWF mutations cannot be found in all VWD cases. Although an increasing number of genetic modifiers have been identified, even more rare genetic variants that impact VWF plasma levels likely remain to be discovered. Here, we summarize recent evidence that modulation of the early secretory pathway has great impact on the biogenesis and release of WPBs. Based on these findings, we propose that rare, as yet unidentified quantitative trait loci influencing intracellular VWF transport contribute to highly variable VWF levels in the population. These may underlie the thrombotic complications linked to high VWF levels, as well as the bleeding tendency in individuals with low VWF levels.
- Published
- 2022
21. Osteoprotegerin modulates platelet adhesion to von Willebrand factor during release from endothelial cells
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Nikolett Wohner, Peter J. Lenting, Bas de Laat, Philip G de Groot, Vincent Muczynski, Silvie Sebastian, Dana Huskens, RS: Carim - B01 Blood proteins & engineering, and Biochemie
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congenital, hereditary, and neonatal diseases and abnormalities ,blood platelets ,Inflammation ,von Willebrand factor ,Mice ,chemistry.chemical_compound ,Platelet Adhesiveness ,Von Willebrand factor ,Osteoprotegerin ,hemic and lymphatic diseases ,medicine ,WEIBEL-PALADE BODIES ,Animals ,Humans ,Platelet ,Secretion ,THROMBOTIC THROMBOCYTOPENIC PURPURA ,Ristocetin ,biology ,Chemistry ,Hematology ,endothelial cells ,Cell biology ,IN-VIVO SURVIVAL ,MODEL ,von Willebrand Diseases ,Concanavalin A ,inflammation ,osteoprotegerin ,cardiovascular system ,biology.protein ,SECRETION ,medicine.symptom ,circulatory and respiratory physiology ,Binding domain - Abstract
Background Platelet-binding Von Willebrand Factor (VWF) strings assemble upon stimulated secretion from endothelial cells. Objectives To investigate the efficiency of platelet binding to multi-molecular VWF bundles secreted from endothelial cells and to investigate the role of osteoprotegerin, a protein located in Weibel-Palade bodies that interacts with the VWF platelet binding domain. Methods The nanobody VWF/AU-a11 that specifically binds to VWF in its active platelet-binding conformation was used to investigate the conformation of VWF. Results Upon stimulated secretion from endothelial cells, VWF strings were only partially covered with platelets, while a VWD-type 2B mutation or ristocetin enhanced platelet binding by 2-3-fold. Osteoprotegrin, reduces platelet adhesion to VWF by 40±18% in perfusion assays. siRNA-mediated down-regulation of endothelial osteoprotegerin expression resulted in a 1.8-fold increase in platelet adhesion to VWF strings. Upon viral infection, there is a concordant rise in VWF and osteoprotegerin plasma levels. Unexpectedly, no such increase was observed in plasma of desmopressin-treated hemophilia A-patients. In a mouse model, osteoprotegerin expression was low in liver endothelial cells of vehicle-treated mice, and concanavalin A-treatment increased VWF and osteoprotegerin expression 4- and 40-fold, respectively. This increase was translated in a 30-fold increased osteoprotegerin/VWF ratio in plasma. Conclusions Release of VWF from endothelial cells opens the platelet-binding site, irrespective of the presence of flow. However, not all available platelet-binding sites are being occupied, suggesting some extent of regulation. Part of this regulation involves endothelial proteins that are co-secreted with VWF, like osteoprotegerin. This regulatory mechanism may be of more relevance under inflammatory conditions.
- Published
- 2022
22. Multifaceted pathomolecular mechanism of a VWF large deletion involved in the pathogenesis of severe VWD
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Ulrich Budde, Muhammad Ahmer Jamil, Orla Rawley, N. Marquardt, Hamideh Yadegari, David Lillicrap, Johannes Oldenburg, Jens Müller, and Osman El-Maarri
- Subjects
Weibel-Palade Bodies ,biology ,Angiogenesis ,Cellular differentiation ,Endothelial Cells ,Blood vessel morphogenesis ,Hematology ,medicine.disease ,Cell biology ,Endothelial stem cell ,P-Selectin ,Phenotype ,Von Willebrand factor ,hemic and lymphatic diseases ,von Willebrand Factor ,Von Willebrand disease ,medicine ,biology.protein ,Humans ,Interleukin 8 ,Cell adhesion - Abstract
An in-frame heterozygous large deletion of exons 4 through 34 of the von Willebrand factor (VWF) gene was identified in a type 3 von Willebrand disease (VWD) index patient (IP), as the only VWF variant. The IP exhibited severe bleeding episodes despite prophylaxis treatment, with a short VWF half-life after infusion of VWF/factor VIII concentrates. Transcript analysis confirmed transcription of normal VWF messenger RNA besides an aberrant deleted transcript. The IP endothelial colony-forming cells (ECFCs) exhibited a defect in the VWF multimers and Weibel-Palade bodies (WPBs) biogenesis, although demonstrating normal VWF secretion compared with healthy cells. Immunostaining of IP-ECFCs revealed subcellular mislocalization of WPBs pro-inflammatory cargos angiopoietin-2 (Ang2, nuclear accumulation) and P-selectin. Besides, the RNA-sequencing (RNA-seq) analysis showed upregulation of pro-inflammatory and proangiogenic genes, P-selectin, interleukin 8 (IL-8), IL-6, and GROα, copackaged with VWF into WPBs. Further, whole-transcriptome RNA-seq and subsequent gene ontology (GO) enrichment analysis indicated the most enriched GO-biological process terms among the differentially expressed genes in IP-ECFCs were regulation of cell differentiation, cell adhesion, leukocyte adhesion to vascular endothelial, blood vessel morphogenesis, and angiogenesis, which resemble downstream signaling pathways associated with inflammatory stimuli and Ang2 priming. Accordingly, our functional experiments exhibited an increased endothelial cell adhesiveness and interruption in endothelial cell–cell junctions of the IP-ECFCs. In conclusion, the deleted VWF has a dominant-negative impact on multimer assembly and the biogenesis of WPBs, leading to altered trafficking of their pro-inflammatory cargos uniquely, which, in turn, causes changes in cellular signaling pathways, phenotype, and function of the endothelial cells.
- Published
- 2022
23. Syntaxin 5 determines Weibel-Palade body size and von Willebrand factor secretion by controlling Golgi architecture
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Marije Kat, Ellie Karampini, Arie Johan Hoogendijk, Petra Bürgisser, Aat A. Mulder, Floris van Alphen, Jenny Olins, Dirk Geerts, Maartje van den Biggelaar, Coert Margadant, Jan Voorberg, Ruben Bierings, Hematology, Medical oncology laboratory, Experimental Vascular Medicine, Landsteiner Laboratory, and ACS - Microcirculation
- Subjects
Weibel-Palade Bodies ,Qa-SNARE Proteins ,hemic and lymphatic diseases ,von Willebrand Factor ,Body Size ,Endothelial Cells ,Humans ,Hematology ,Cells, Cultured ,Exocytosis - Abstract
Von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells (ECs). VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPBs), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde ER-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometrybased approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in ECs using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted ECs exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion-incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. Taken together, our study has identified SNARE protein STX5 as a novel regulator of WPB biogenesis.
- Published
- 2022
24. Spire1 and Myosin Vc promote Ca2+-evoked externalization of von Willebrand factor in endothelial cells
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Holthenrich, Anna, Terglane, Julian, Naß, Johannes, Mietkowska, Magdalena, Kerkhoff, Eugen, and Gerke, Volker
- Published
- 2022
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25. Involvement of Weibel-Palade bodies in Kaposiform haemangioendothelioma.
- Author
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Matsunaga T, Yamada N, Yoshida Y, Yoshida A, Nakahara T, and Yamamoto O
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- Humans, Weibel-Palade Bodies, Kasabach-Merritt Syndrome, Hemangioendothelioma, Sarcoma, Kaposi
- Published
- 2023
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26. Angiopoietin-2 binds to multiple interactive sites within von Willebrand factor.
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Texier A, Lenting PJ, Denis CV, Roullet S, and Christophe OD
- Abstract
Background: Biosynthesis of von Willebrand factor (VWF) in endothelial cells drives the formation of storage-organelles known as Weibel-Palade bodies (WPBs). WPBs also contain several other proteins, including angiopoietin-2 (Ang-2)., Objectives: At present, the molecular basis of the VWF-Ang-2 interaction is poorly understood. Here, we used immunosorbent-binding assays and specific recombinant VWF fragments to analyze VWF-Ang-2 interactions., Results: We found that VWF bound to immobilized Ang-2 most efficiently (half-maximal binding at 0.5 ± 0.1 μg/mL) under conditions of high CaCl
2 (10 mM) and slightly acidic pH (6.4-7.0). Interestingly, several isolated recombinant VWF domains (A1/Fc, A2/Fc, D4/Fc, and D'D3-HPC4) displayed dose-dependent binding to immobilized Ang-2. Binding appeared specific, as antibodies against D'D3, A1, and A2 significantly reduced the binding of these domains to Ang-2. Complexes between VWF and Ang-2 in plasma could be detected by immunoprecipitation- and immunosorbent assays. Unexpectedly, control experiments also revealed complexes between VWF and angiopoietin-1 (Ang-1), a protein structurally homologous to Ang-2. Furthermore, direct binding studies showed dose-dependent binding of VWF to immobilized Ang-1 (half-maximal binding at 1.8 ± 1.0 μg/mL). Interestingly, rather than competing for Ang-1 binding, Ang-2 enhanced the binding of VWF to Ang-1 about 3-fold. Competition experiments further revealed that binding to VWF does not prevent Ang-1 and Ang-2 from binding to Tie-2., Conclusion: Our data show that both Ang-1 and Ang-2 bind to VWF, seemingly using different interactive sites. Ang-2 modulates the binding of VWF to Ang-1, the (patho)-physiological consequences of which remain to be investigated., (© 2023 The Authors.)- Published
- 2023
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27. The telopode- and filopode-projecting heterogeneous stromal cells of the human sclera niche.
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Petrea, C.E., Vrapciu, A.D., Rusu, M.C., Crăiţoiu, Ş., and Mănoiu, V.S.
- Subjects
STROMAL cells ,SCLERA ,STEM cells ,PERILIPIN ,PHENOTYPES ,TRANSMISSION electron microscopy - Abstract
Telocytes (TCs) are stromal cells defined by the presence of long and slender prolongations (telopodes). They are a biologically and functionally heterogeneous population that has not been previously investigated in the sclera. The purpose of this study is to investigate the presence and characteristics of scleral telocytes through a combined immunohistochemical and transmission electron microscopy (TEM) study using samples from ten adult patients. Stromal cells with a TC-like morphology expressed CD34, CD45, CD105, vimentin and occasionally CD68 but were negative for collagen III, CD31, CD133, and CD146. Conjunctival epithelial cells expressed CD45, CD105, CD146, and vimentin. These phenotypes support a scleral niche with immune TCs and haematopoietic stem cells (HSCs). In TEM, we often found spindle-shaped stromal cells projecting telopodes or filopodes, with extremely long nuclei extended even within those prolongations. We separated these cells into a light subtype, which contained a complete set of organelles, and a dark subtype, consisting of undifferentiated stem/progenitor cells. The light cells contained dense vesicles, Weibel–Palade bodies, and rounded α-granule-like structures. These storage areas for the von Willebrand factor (vWF) are known to express selectins that are critically involved in HSC homing and could also indicate endothelial progenitors. The dark cells were scarcely myoid, populated the episcleral perivascular niches and the scleral stroma, and were equipped with lipid storage areas such as lamellar bodies and lipid droplets (LDs). Previously, unreported intranuclear LDs were found in these cells, which is characteristic of an HSC population. It appears that the human scleral stroma is a niche harbouring TC-like cells with immune and HSC phenotypes, and the mere presence or characteristics of telopodes are not enough to differentiate them. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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28. Omega-3 Fatty Acids Modulate Weibel-Palade Body Degranulation and Actin Cytoskeleton Rearrangement in PMA-Stimulated Human Umbilical Vein Endothelial Cells
- Author
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Corinna S. Bürgin-Maunder, Peter R. Brooks, and Fraser D. Russell
- Subjects
omega-3 fatty acids ,von willebrand factor ,weibel-palade bodies ,endothelial function ,actin cytoskeleton ,Biology (General) ,QH301-705.5 - Abstract
Long chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs) produce cardiovascular benefits by improving endothelial function. Endothelial cells store von Willebrand factor (vWF) in cytoplasmic Weibel-Palade bodies (WPBs). We examined whether LC n-3 PUFAs regulate WPB degranulation using cultured human umbilical vein endothelial cells (HUVECs). HUVECs were incubated with or without 75 or 120 µM docosahexaenoic acid or eicosapentaenoic acid for 5 days at 37 °C. WPB degranulation was stimulated using phorbol 12-myristate 13-acetate (PMA), and this was assessed by immunocytochemical staining for vWF. Actin reorganization was determined using phalloidin-TRITC staining. We found that PMA stimulated WPB degranulation, and that this was significantly reduced by prior incubation of cells with LC n-3 PUFAs. In these cells, WPBs had rounded rather than rod-shaped morphology and localized to the perinuclear region, suggesting interference with cytoskeletal remodeling that is necessary for complete WPB degranulation. In line with this, actin rearrangement was altered in cells containing perinuclear WPBs, where cells exhibited a thickened actin rim in the absence of prominent cytoplasmic stress fibers. These findings indicate that LC n-3 PUFAs provide some protection against WBP degranulation, and may contribute to an improved understanding of the anti-thrombotic effects previously attributed to LC n-3 PUFAs.
- Published
- 2013
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29. The Use of Fluorescently Labeled ARC1779 Aptamer for Assessing the Effect of H2O2 on von Willebrand Factor Exocytosis
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Nikolay V. Goncharov, Elena Yu. Rybakova, P. P. Avdonin, Sergey K. Trufanov, Pavel V. Avdonin, and Aleksandra A. Tsitrina
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030204 cardiovascular system & hematology ,Biochemistry ,Exocytosis ,Umbilical vein ,03 medical and health sciences ,0302 clinical medicine ,Thrombin ,Von Willebrand factor ,von Willebrand Factor ,Human Umbilical Vein Endothelial Cells ,medicine ,Fluorescence microscope ,Humans ,Secretion ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Reactive oxygen species ,Weibel-Palade Bodies ,biology ,Hydrogen Peroxide ,General Medicine ,Aptamers, Nucleotide ,chemistry ,Second messenger system ,cardiovascular system ,Biophysics ,biology.protein ,medicine.drug - Abstract
Abstract Here, we propose a new approach for quantitative estimation of von Willebrand factor (vWF) exposed on the surface of endothelial cells (ECs) using the ARC1779 aptamer that interacts with the vWF A1 domain. To visualize complex formation between vWF and the aptamer, the latter was conjugated with the Cy5 fluorescent label. Cultured human umbilical vein endothelial cells (HUVEC) were stained with the ARC1779-Cy5 conjugate and imaged with a fluorescence microscope. The images were analyzed with the CellProfiler software. vWF released from the Weibel–Palade bodies was observed as bright dot-like structures of round and irregular shape, the number of which increased several times after HUVEC exposure to histamine or thrombin. Staining with ARC1779-Cy5 also revealed long filamentous vWF structures on the surface of activated HUVEC. vWF secretion by ECs is activated by the second messengers cAMP and Ca2+. There is evidence that hydrogen peroxide also acts as a second messenger in ECs. In addition, exogenous H2O2 formed in leukocytes can enter ECs. The aim of our study was to determine the effect of H2O2 on the vWF exposure at the surface of HUVEC using the proposed method. It was shown that hydrogen peroxide at concentration 100 µM, which is lower than the cytotoxicity threshold of H2O2 for cultured HUVEC, increased several times the number of dot-like structures and total amount of vWF exposed on plasma membrane of HUVEC, which suggest that H2O2 acts as a mediator that activates exocytosis of Weibel–Palade bodies and vWF secretion in the vascular endothelium during inflammation and upon elevated generation of endogenous reactive oxygen species in ECs.
- Published
- 2020
30. VWF maturation and release are controlled by 2 regulators of Weibel-Palade body biogenesis: exocyst and BLOC-2
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Chao Fang, Anish V. Sharda, Ionita Ghiran, Joshua Harrison, Joseph E. Italiano, Adrian R. Wilkie, Lourdes M. Mendez, Alexandra M. Barr, and Robert Flaumenhaft
- Subjects
Limonins ,0301 basic medicine ,Immunoprecipitation ,Endosome ,Immunology ,Exocyst ,Endosomes ,Biochemistry ,Exocytosis ,03 medical and health sciences ,0302 clinical medicine ,hemic and lymphatic diseases ,von Willebrand Factor ,Human Umbilical Vein Endothelial Cells ,Weibel–Palade body ,Humans ,Platelet ,Secretion ,Weibel-Palade Bodies ,Chemistry ,Intracellular Signaling Peptides and Proteins ,Cell Biology ,Hematology ,Cell biology ,030104 developmental biology ,030220 oncology & carcinogenesis ,Biogenesis - Abstract
von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized in endothelial cells and stored in Weibel-Palade bodies (WPBs). Understanding the mechanisms underlying WPB biogenesis and exocytosis could enable therapeutic modulation of endogenous VWF, yet optimal targets for modulating VWF release have not been established. Because biogenesis of lysosomal related organelle-2 (BLOC-2) functions in the biogenesis of platelet dense granules and melanosomes, which like WPBs are lysosome-related organelles, we hypothesized that BLOC-2–dependent endolysosomal trafficking is essential for WPB biogenesis and sought to identify BLOC-2–interacting proteins. Depletion of BLOC-2 caused misdirection of cargo-carrying transport tubules from endosomes, resulting in immature WPBs that lack endosomal input. Immunoprecipitation of BLOC-2 identified the exocyst complex as a binding partner. Depletion of the exocyst complex phenocopied BLOC-2 depletion, resulting in immature WPBs. Furthermore, releasates of immature WPBs from either BLOC-2 or exocyst-depleted endothelial cells lacked high-molecular weight (HMW) forms of VWF, demonstrating the importance of BLOC-2/exocyst-mediated endosomal input during VWF maturation. However, BLOC-2 and exocyst showed very different effects on VWF release. Although BLOC-2 depletion impaired exocytosis, exocyst depletion augmented WPB exocytosis, indicating that it acts as a clamp. Exposure of endothelial cells to a small molecule inhibitor of exocyst, Endosidin2, reversibly augmented secretion of mature WPBs containing HMW forms of VWF. These studies show that, although BLOC-2 and exocyst cooperate in WPB formation, only exocyst serves to clamp WPB release. Exocyst function in VWF maturation and release are separable, a feature that can be exploited to enhance VWF release.
- Published
- 2020
31. Tip-end fusion of a rod-shaped secretory organelle
- Author
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Johannes Naß, Sophia N. Koerdt, Anja Biesemann, Tarek Chehab, Takao Yasuda, Mitsunori Fukuda, Fernando Martín-Belmonte, and Volker Gerke
- Subjects
Pharmacology ,Cellular and Molecular Neuroscience ,Weibel-Palade Bodies ,von Willebrand Factor ,Human Umbilical Vein Endothelial Cells ,Molecular Medicine ,Humans ,Cell Biology ,Molecular Biology ,Cells, Cultured ,Exocytosis - Abstract
Weibel–Palade bodies (WPB) are elongated, rod-like secretory organelles unique to endothelial cells that store the pro-coagulant von-Willebrand factor (VWF) and undergo regulated exocytosis upon stimulation with Ca2+- or cAMP-raising agonists. We show here that WPB preferentially initiate fusion with the plasma membrane at their tips and identify synaptotagmin-like protein 2-a (Slp2-a) as a positive regulator of VWF secretion most likely mediating this topological selectivity. Following secretagogue stimulation, Slp2-a accumulates at one WPB tip before fusion occurs at this site. Depletion of Slp2-a reduces Ca2+-dependent secretion of highly multimeric VWF and interferes with the formation of actin rings at WPB–plasma membrane fusion sites that support the expulsion of the VWF multimers and most likely require a tip-end fusion topology. Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] binding via the C2A domain of Slp2-a is required for accumulation of Slp2-a at the tip ends of fusing WPB, suggesting that Slp2-a mediates polar exocytosis by initiating contacts between WPB tips and plasma membrane PI(4,5)P2.
- Published
- 2022
32. Acidification of endothelial Weibel-Palade bodies is mediated by the vacuolar-type H + - ATPase
- Author
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Julian, Terglane, Dirk, Menche, and Volker, Gerke
- Subjects
Vacuolar Proton-Translocating ATPases ,Multidisciplinary ,Weibel-Palade Bodies ,von Willebrand Factor ,Endothelial Cells ,Hydrogen-Ion Concentration ,Cells, Cultured ,Exocytosis - Abstract
Weibel-Palade bodies (WPB) are unique secretory granules of endothelial cells that store the procoagulant von-Willebrand factor (VWF) in a highly compacted form. Upon exocytosis the densely packed VWF unfurls into long strands that expose binding sites for circulating platelets and thereby initiate the formation of a platelet plug at sites of blood vessel injury. Dense packing of VWF requires the establishment of an acidic pH in the lumen of maturing WPB but the mechanism responsible for this acidification has not yet been fully established. We show here that subunits of the vacuolar-type H+-ATPase are present on mature WPB and that interference with the proton pump activity of the ATPase employing inhibitors of different chemical nature blocks proper acidification and maturation of WPB. Furthermore, depletion of the V-ATPase subunit V0d1 from primary endothelial cells prevents WPB acidification and the establishment of an elongated morphology of WPB that is dictated by the densely packed VWF tubules. Thus, the vacuolar-type H+-ATPase present on WPB is required for proper acidification and maturation of the organelle.
- Published
- 2022
33. von Willebrand factor binds to angiopoietin-2 within endothelial cells and after release from Weibel-Palade bodies.
- Author
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Mobayen G, Smith K, Ediriwickrema K, Starke RD, Solomonidis EG, Laffan MA, Randi AM, and McKinnon TAJ
- Subjects
- Humans, Angiopoietin-2 metabolism, Exocytosis, Human Umbilical Vein Endothelial Cells metabolism, Cells, Cultured, von Willebrand Factor metabolism, Weibel-Palade Bodies metabolism
- Abstract
Background: The von Willebrand factor (VWF) is a multimeric plasma glycoprotein essential for hemostasis, inflammation, and angiogenesis. The majority of VWF is synthesized by endothelial cells (ECs) and stored in Weibel-Palade bodies (WPB). Among the range of proteins shown to co-localize to WPB is angiopoietin-2 (Angpt-2), a ligand of the receptor tyrosine kinase Tie-2. We have previously shown that VWF itself regulates angiogenesis, raising the hypothesis that some of the angiogenic activity of VWF may be mediated by its interaction with Angpt-2., Methods: Static-binding assays were used to probe the interaction between Angpt-2 and VWF. Binding in media from cultured human umbilical vein ECs s and in plasma was determined by immunoprecipitation experiments. Immunofluorescence was used to detect the presence of Angpt-2 on VWF strings, and flow assays were used to investigate the effect on VWF function., Results: Static-binding assays revealed that Angpt-2 bound to VWF with high affinity (K
D,app ∼3 nM) in a pH and calcium-dependent manner. The interaction was localized to the VWF A1 domain. Co-immunoprecipitation experiments demonstrated that the complex persisted following stimulated secretion from ECs and was present in plasma. Angpt-2 was also visible on VWF strings on stimulated ECs. The VWF-Angpt-2 complex did not inhibit the binding of Angpt-2 to Tie-2 and did not significantly interfere with VWF-platelet capture., Conclusions: Together, these data demonstrate a direct binding interaction between Angpt-2 and VWF that persists after secretion. VWF may act to localize Angpt-2; further work is required to establish the functional consequences of this interaction., Competing Interests: Declaration of competing interests The authors declare no competing financial interests., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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34. NOTCH inhibition promotes bronchial stem cell renewal and epithelial barrier integrity after irradiation
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Ludwig Dubois, Marc Vooijs, Arjan J. Groot, Eloy Moreno Roig, Annette van den Berg, Carolien Wansleeben, Lorena Giuranno, MUMC+: MA Radiotherapie OC (9), RS: GROW - R2 - Basic and Translational Cancer Biology, and Radiotherapie
- Subjects
0301 basic medicine ,Angiogenesis ,Apoptosis ,von Willebrand factor ,radiation‐induced lung injury ,ANGIOGENESIS ,Cell therapy ,0302 clinical medicine ,Tissue‐specific Progenitor and Stem Cells ,Weibel-Palade bodies ,HUMAN IPSCS ,PLASTICITY ,lcsh:R5-920 ,Receptors, Notch ,Chemistry ,lcsh:Cytology ,Cell Differentiation ,General Medicine ,Lung Injury ,glycolysis ,air‐liquid interface system ,medicine.anatomical_structure ,DIFFERENTIATION ,SECRETION ,Stem cell ,lcsh:Medicine (General) ,Signal Transduction ,STORAGE ,VON-WILLEBRAND-FACTOR ,Notch signaling pathway ,induced pluripotent stem cell-derived endothelial cells ,METABOLISM ,03 medical and health sciences ,In vivo ,medicine ,Humans ,primary bronchial epithelial cells ,lcsh:QH573-671 ,radiotherapy ,Cell Proliferation ,Epithelial Cells ,Cell Biology ,Epithelium ,030104 developmental biology ,ANGIOPOIETIN-2 ,Cancer research ,Respiratory epithelium ,NOTCH signaling ,cell therapy ,030217 neurology & neurosurgery ,Ex vivo ,Developmental Biology ,GENERATION - Abstract
Hyperactivity of the NOTCH pathway is associated with tumor growth and radiotherapy resistance in lung cancer, and NOTCH/γ-secretase inhibitors (GSIs) are a potential therapeutic target. The therapeutic outcome, however, is often restricted by the dose-limiting toxicity of combined treatments on the surrounding healthy tissue. The NOTCH signaling pathway is also crucial for homeostasis and repair of the normal airway epithelium. The effects of NOTCH/γ-secretase inhibition on the irradiation of normal lung epithelium are unknown and may counteract antitumor activity. Here we, therefore, investigated whether normal tissue toxicity to radiation is altered upon NOTCH pathway inhibition. We established air-liquid interface pseudostratified and polarized cultures from primary human bronchial epithelial cells and blocked NOTCH signaling alone or after irradiation with small-molecule NOTCH inhibitor/GSI. We found that the reduction in proliferation and viability of bronchial stem cells (TP63+) in response to irradiation is rescued with concomitant NOTCH inhibition. This correlated with reduced activation of the DNA damage response and accelerated repair by 24 hours and 3 days postirradiation. The increase in basal cell proliferation and viability in GSI-treated and irradiated cultures resulted in an improved epithelial barrier function. Comparable results were obtained after in vivo irradiation, where the combination of NOTCH inhibition and irradiation increased the percentage of stem cells and ciliated cells ex vivo. These encourage further use of normal patient tissue for toxicity screening of combination treatments and disclose novel interactions between NOTCH inhibition and radiotherapy and opportunities for tissue repair after radiotherapy. Significance statement Radiation-induced lung injury is a dose-limiting toxicity that limits the effective dose that should be administered and forces the interruption of the treatment. The NOTCH signaling pathway is a potential therapeutic target for lung cancer because its inhibition reduces tumor growth and synergizes with radiotherapy and chemotherapy in preclinical models. However, the effect of inhibiting NOTCH in irradiated normal lung tissue is not known and could impact the therapeutic benefit of combination treatments. This study demonstrates that small-molecule inhibitors of the NOTCH pathway enhance the survival of irradiated primary human and murine bronchial epithelial lung stem cells. This finding may be beneficial in lung cancer treatment with radiotherapy and NOTCH inhibitors by protecting normal lung tissue while increasing tumor control.
- Published
- 2020
35. Characterization of large in-frame von Willebrand factor deletions highlights differing pathogenic mechanisms
- Author
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Cartwright, A., Webster, S.J., Jong, A. de, Dirven, R.J., Bloomer, L.D.S., AL-Buhairan, A.M., Budde, U., Hallden, C., Habart, D., Goudemand, J., Peake, I.R., Eikenboom, J.C.J., Goodeve, A.C., Hampshire, D.J., European Grp von Willebrand Dis, and Willebrand Dis Study Grp von
- Subjects
0301 basic medicine ,DNA Copy Number Variations ,030204 cardiovascular system & hematology ,von Willebrand Disease, Type 1 ,Thrombosis and Hemostasis ,law.invention ,Pathogenesis ,03 medical and health sciences ,Exon ,0302 clinical medicine ,Von Willebrand factor ,law ,hemic and lymphatic diseases ,von Willebrand Factor ,Von Willebrand disease ,medicine ,Humans ,Secretion ,Copy-number variation ,Weibel-Palade Bodies ,biology ,Hematology ,medicine.disease ,Molecular biology ,In vitro ,von Willebrand Diseases ,030104 developmental biology ,biology.protein ,Recombinant DNA - Abstract
Copy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion.
- Published
- 2020
36. Orchestration of Primary Hemostasis by Platelet and Endothelial Lysosome-Related Organelles
- Author
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Ruben Bierings, Jan Voorberg, and Ellie Karampini
- Subjects
Blood Platelets ,0301 basic medicine ,von Willebrand factor ,030204 cardiovascular system & hematology ,Cytoplasmic Granules ,03 medical and health sciences ,0302 clinical medicine ,Von Willebrand factor ,megakaryocytes ,Organelle ,Humans ,Secretion ,Platelet ,Transcription factor ,Melanosome ,Weibel-Palade Bodies ,biology ,Chemistry ,Blood Coagulation Disorders ,endothelial cells ,Cell biology ,lysosome-related organelles ,030104 developmental biology ,Hemostasis ,platelets ,hemostasis ,biology.protein ,Collagen ,Lysosomes ,Cardiology and Cardiovascular Medicine ,Biogenesis - Abstract
Megakaryocyte-derived platelets and endothelial cells store their hemostatic cargo in α- and δ-granules and Weibel-Palade bodies, respectively. These storage granules belong to the lysosome-related organelles (LROs), a heterogeneous group of organelles that are rapidly released following agonist-induced triggering of intracellular signaling pathways. Following vascular injury, endothelial Weibel-Palade bodies release their content into the vascular lumen and promote the formation of long VWF (von Willebrand factor) strings that form an adhesive platform for platelets. Binding to VWF strings as well as exposed subendothelial collagen activates platelets resulting in the release of α- and δ-granules, which are crucial events in formation of a primary hemostatic plug. Biogenesis and secretion of these LROs are pivotal for the maintenance of proper hemostasis. Several bleeding disorders have been linked to abnormal generation of LROs in megakaryocytes and endothelial cells. Recent reviews have emphasized common pathways in the biogenesis and biological properties of LROs, focusing mainly on melanosomes. Despite many similarities, LROs in platelet and endothelial cells clearly possess distinct properties that allow them to provide a highly coordinated and synergistic contribution to primary hemostasis by sequentially releasing hemostatic cargo. In this brief review, we discuss in depth the known regulators of α- and δ-granules in megakaryocytes/platelets and Weibel-Palade bodies in endothelial cells, starting from transcription factors that have been associated with granule formation to protein complexes that promote granule maturation. In addition, we provide a detailed view on the interplay between platelet and endothelial LROs in controlling hemostasis as well as their dysfunction in LRO related bleeding disorders.
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- 2020
37. Differential Osteoprotegerin Kinetics after Stimulation with Desmopressin and Lipopolysaccharides In Vivo
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Hubert Hayden, Bernd Jilma, Christian Schoergenhofer, Ulla Derhaschnig, Christa Firbas, Katarina D. Kovacevic, Michael Schwameis, Christine Brostjan, and Nina Buchtele
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Lipopolysaccharides ,musculoskeletal diseases ,0301 basic medicine ,medicine.medical_specialty ,Pilot Projects ,Stimulation ,030204 cardiovascular system & hematology ,Placebo ,03 medical and health sciences ,0302 clinical medicine ,Double-Blind Method ,Von Willebrand factor ,Osteoprotegerin ,In vivo ,Internal medicine ,von Willebrand Factor ,medicine ,Humans ,Deamino Arginine Vasopressin ,Desmopressin ,Cross-Over Studies ,Weibel-Palade Bodies ,biology ,business.industry ,Endothelial Cells ,Hematology ,Healthy Volunteers ,Kinetics ,030104 developmental biology ,Endocrinology ,medicine.anatomical_structure ,Austria ,biology.protein ,business ,Biomarkers ,hormones, hormone substitutes, and hormone antagonists ,Blood sampling ,medicine.drug ,Blood vessel - Abstract
Osteoprotegerin (OPG) regulates bone metabolism by reducing the activation of osteoclasts, but may also be involved in blood vessel calcification and atherosclerosis. Within endothelial cells OPG is stored in Weibel–Palade bodies (WPBs). Blood kinetics of OPG are essentially unknown. We aimed to assess these using two distinct in vivo models; one after stimulation with desmopressin (DDAVP) and another after stimulation with lipopolysaccharide (LPS). Both clinical trials were conducted at the Department of Clinical Pharmacology at the Medical University of Vienna, Austria. Participants received desmopressin (0.3 µg/kg), LPS (2 ng/kg), or placebo (sodium chloride 0.9%) with subsequent blood sampling at time points up to 24 hours after administration. The primary objective of this study was to investigate the plasma kinetics of OPG after stimulation with desmopressin and LPS. Secondary analyses included the release of other WPB contents including von Willebrand factor (vWF). This analysis included 31 healthy volunteers (n = 16 for desmopressin and placebo, n = 15 for LPS). Infusion of desmopressin did not increase OPG concentrations compared with placebo, while LPS infusion significantly increased OPG levels, both compared with desmopressin (p
- Published
- 2020
38. Sec22b determines Weibel-Palade body length by controlling anterograde ER-Golgi transport
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Petra E. Bürgisser, Jan Voorberg, Aat A. Mulder, Ruben Bierings, Dirk Geerts, Ellie Karampini, Jenny Olins, Carolina R. Jost, and Hematology laboratory
- Subjects
030204 cardiovascular system & hematology ,Exocytosis ,Article ,03 medical and health sciences ,symbols.namesake ,0302 clinical medicine ,Von Willebrand factor ,hemic and lymphatic diseases ,von Willebrand Factor ,Organelle ,Weibel–Palade body ,Secretion ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,Weibel-Palade Bodies ,biology ,Chemistry ,Endoplasmic reticulum ,Endothelial Cells ,Hematology ,Golgi apparatus ,Cell biology ,cardiovascular system ,Axoplasmic transport ,symbols ,biology.protein ,circulatory and respiratory physiology - Abstract
Von Willebrand factor (VWF) is a multimeric hemostatic protein that is synthesized in endothelial cells, where it is stored for secretion in elongated secretory organelles called Weibel-Palade bodies (WPB). The hemostatic activity of VWF is strongly related to the length of these bodies, but how endothelial cells control the dimensions of their WPB is unclear. In this study, using a targeted short hairpin RNA screen, we identified longin-SNARE Sec22b as a novel determinant of WPB size and VWF trafficking. We found that Sec22b depletion resulted in loss of the typically elongated WPB morphology together with disintegration of the Golgi and dilation of rough endoplasmic reticulum cisternae. This was accompanied by reduced proteolytic processing of VWF, accumulation of VWF in the dilated rough endoplasmic reticulum and reduced basal and stimulated VWF secretion. Our data demonstrate that the elongation of WPB, and thus adhesive activity of their cargo VWF, is determined by the rate of anterograde transport between endoplasmic reticulum and Golgi, which depends on Sec22b-containing SNARE complexes.
- Published
- 2020
39. Spire1 and Myosin Vc promote Ca2+-evoked externalization of von Willebrand factor in endothelial cells
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Anna Holthenrich, Julian Terglane, Johannes Naß, Magdalena Mietkowska, Eugen Kerkhoff, and Volker Gerke
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Pharmacology ,Actin nucleation ,Microscopy, Confocal ,Weibel-Palade Bodies ,Endothelial activation ,Blotting, Western ,Green Fluorescent Proteins ,Microfilament Proteins ,Myosin Type V ,Calcium signaling ,Nuclear Proteins ,Cell Biology ,Models, Biological ,Exocytosis ,Cellular and Molecular Neuroscience ,Weibel–Palade bodies ,von Willebrand Factor ,Human Umbilical Vein Endothelial Cells ,Humans ,Molecular Medicine ,Original Article ,Calcium ,RNA Interference ,Molecular Biology ,Cells, Cultured - Abstract
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. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-021-04108-x.
- Published
- 2022
40. Is Endothelial Activation a Critical Event in Thrombotic Thrombocytopenic Purpura?
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Raphael Cauchois, Romain Muller, Marie Lagarde, Françoise Dignat-George, Edwige Tellier, Gilles Kaplanski, Centre recherche en CardioVasculaire et Nutrition = Center for CardioVascular and Nutrition research (C2VN), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Service de médecine interne et immunologie clinique, AP-HM, Marseille, Centre national de référence des microangiopathies thrombotiques (CNR-MAT), Laboratoire d’Hématologie [AP-HM Hôpital de la Timone], Hôpital de la Timone [CHU - APHM] (TIMONE), and Centre national de référence des microangiopathies (CNR-MAT)
- Subjects
Thrombotic thrombocytopenic purpura ,[SDV]Life Sciences [q-bio] ,Weibel-Palade bodies ,General Medicine ,endothelial cells - Abstract
International audience; Thrombotic thrombocytopenic purpura (TTP) is a severe thrombotic microangiopathy. The current pathophysiologic paradigm suggests that the ADAMTS13 deficiency leads to Ultra Large-Von Willebrand Factor multimers accumulation with generation of disseminated microthrombi. Nevertheless, the role of endothelial cells in this pathology remains an issue. In this review, we discuss the various clinical, in vitro and in vivo experimental data that support the important role of the endothelium in this pathology, suggesting that ADAMTS13 deficiency may be a necessary but not sufficient condition to induce TTP. The "second hit" model suggests that in TTP, in addition to ADAMTS13 deficiency, endogenous or exogenous factors induce endothelial activation affecting mainly microvascular cells. This leads to Weibel-Palade bodies degranulation, resulting in UL-VWF accumulation in microcirculation. This endothelial activation seems to be worsened by various amplification loops, such as the complement system, nucleosomes and free heme.
- Published
- 2023
41. V-ATPase V0a1 promotes Weibel–Palade body biogenesis through the regulation of membrane fission
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Yuka Eura, Yasuo Yamazaki, and Koichi Kokame
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Vacuolar Proton-Translocating ATPases ,Organelle Biogenesis ,General Immunology and Microbiology ,Weibel-Palade Bodies ,QH301-705.5 ,General Neuroscience ,membrane transport ,membrane fission ,Science ,Cell Membrane ,organellegenesis ,Endothelial Cells ,General Medicine ,Cell Biology ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,hemostasis ,Humans ,Medicine ,Biology (General) ,Research Article ,Human - Abstract
Membrane fission, the division of a membrane-bound structure into two discrete compartments, is essential for diverse cellular events, such as endocytosis and vesicle/granule biogenesis; however, the process remains unclear. The hemostatic protein von Willebrand factor is produced in vascular endothelial cells and packaged into specialized secretory granules, Weibel–Palade bodies (WPBs) at the trans-Golgi network (TGN). Here, we reported that V0a1, a V-ATPase component, is required for the membrane fission of WPBs. We identified two V0a isoforms in distinct populations of WPBs in cultured endothelial cells, V0a1 and V0a2, on mature and nascent WPBs, respectively. Although WPB buds were formed, WPBs could not separate from the TGN in the absence of V0a1. Screening using dominant–negative forms of known membrane fission regulators revealed protein kinase D (PKD) as an essential factor in biogenesis of WPBs. Further, we showed that the induction of wild-type PKDs in V0a1-depleted cells does not support the segregation of WPBs from the TGN; suggesting a primary role of V0a1 in the membrane fission of WPBs. The identification of V0a1 as a new membrane fission regulator should facilitate the understanding of molecular events that enable membrane fission.
- Published
- 2021
42. Metabolic regulation of differentiation and maturation
- Author
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Es - Tiemeier, G.L. van, Rabelink, T.J., Berg, B.M. van den, Berg, C.W. van den, Zonneveld, A.J. van, Kooten, C. van, Eikenboom, J., Drukker, M.E., Orlova, V.V., Levi, M., and Leiden University
- Subjects
PGC1-alpha ,iPSC-EC differentiation ,iPSC-derived endothelial cells ,mitochondrial permeability transition pore ,Weibel-Palade bodies ,von Willebrand factor ,cyclosporine A ,glycocalyx ,shear stress ,metabolic switch - Abstract
In this thesis, we addressed the role of cellular metabolism in maturation of hiPSC-ECs. In Chapter 2, we compare hiPSC derived EC functionality and metabolism with primary human microvascular endothelial cells (hMVEC) determining the presence of a luminal glycocalyx as a main functional target. This chapter presents new insights in mitochondrial dysfunction of hiPSC-EC, limiting their ability to produce sufficient glycocalyx and align to shear stress. Underlying the mitochondrial dysfunction, we found that hiPSC-EC have an open mitochondrial permeability transition pore (mPTP), indicating mitochondrial immaturity. By closing the mPTP during differentiation with Cyclosporin-A (CsA), binding to cyclophilin D of the mPTP, we were able to mature mitochondria and improve functionality of these cells, resulting in a reduction in ROS, increased glycocalyx production and the therefore providing iPSC-ECs the ability to align to shear stress. Chapter 3 Continues with the comparison of hiPSC-EC with hMVEC, focusing on von Willebrand Factor (VWF) and the production of Weibel Palade Bodies (WPB). Testing several differentiation protocols and even after addition of CsA, hiPSC-EC were found to lack mature WPBs. We showed that neither shear stress nor co-culture with pericytes could induce WPB formation. By further studying the metabolism with NMR we found that hiPSC-EC have a reduced glycolysis and lactate production and an increased intracellular pH (pHi). This coincides with a reduced expression of the proton coupled monocarboxylate transporter MCT1, which transports H+ and lactate into the cell to keep pHi in balance. Reducing the intracellular pH led to increased presence of functional VWF and maturation of WPB in hiPSC-ECs. In Chapter 4 we addressed the question how hiPSC-EC maintain their redoxbalance and produce enough ATP, since the vast majority of ATP and antioxidants of EC are obtained by glycolysis, which is significantly reduced in iPSC-ECs. We found that hiPSC-EC rely mainly on free fatty acid oxidation and presumably use NADPH do maintain their redox balance. This alternative metabolic state in hiPSCEC was found to be independent of the high expression of the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which regulates fatty acid storage, glucose metabolism, lipid uptake and mitochondrial biogenesis. Since PGC1α is directly activated by ROS and is the master regulator of the energy metabolism, the high levels of PGC1α expression in hiPSCECcould also be a consequence instead of the cause of the observed differences in metabolism. In Chapter 5, we studied the immunogenic surface of hiPSC-ECs, since transplantation induced rejection by the host immune system is an essential hurdle in usage of IPSC derived tissue. Previous studies suggested that IPSC derived cell recognition by the host immune system is diminished compared to transplantation of allogenic human alternatives. On the other hand, endothelial surface MHC class 1 and 2 molecules do play an active, ‘APC like’, role in adult immunity. Therefore, we characterized hiPSC-EC surface immune complexes, unstimulated and after cytokine stimulation. In addition we tested how the observed difference in expression could influence CD8 T-cell activation. Furthermore, we characterized the expression of complement inhibitors on the cell surface, necessary for the protection against unprovoked complement activation in the blood. Chapter 6 provides a summary and discussion of the observations in this thesis, including future perspectives on studying metabolism and metabolepigenetics in iPSC derived kidney organoids by mass spectrometry imaging.
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- 2021
43. [Untitled]
- Author
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Rabelink, T.J., Berg, B.M. van den, Berg, C.W. van den, Zonneveld, A.J. van, Kooten, C. van, Eikenboom, J., Drukker, M.E., Orlova, V.V., Levi, M., and Leiden University
- Subjects
PGC1-alpha ,iPSC-EC differentiation ,iPSC-derived endothelial cells ,mitochondrial permeability transition pore ,Weibel-Palade bodies ,von Willebrand factor ,cyclosporine A ,glycocalyx ,shear stress ,metabolic switch - Abstract
In this thesis, we addressed the role of cellular metabolism in maturation of hiPSC-ECs. In Chapter 2, we compare hiPSC derived EC functionality and metabolism with primary human microvascular endothelial cells (hMVEC) determining the presence of a luminal glycocalyx as a main functional target. This chapter presents new insights in mitochondrial dysfunction of hiPSC-EC, limiting their ability to produce sufficient glycocalyx and align to shear stress. Underlying the mitochondrial dysfunction, we found that hiPSC-EC have an open mitochondrial permeability transition pore (mPTP), indicating mitochondrial immaturity. By closing the mPTP during differentiation with Cyclosporin-A (CsA), binding to cyclophilin D of the mPTP, we were able to mature mitochondria and improve functionality of these cells, resulting in a reduction in ROS, increased glycocalyx production and the therefore providing iPSC-ECs the ability to align to shear stress. Chapter 3 Continues with the comparison of hiPSC-EC with hMVEC, focusing on von Willebrand Factor (VWF) and the production of Weibel Palade Bodies (WPB). Testing several differentiation protocols and even after addition of CsA, hiPSC-EC were found to lack mature WPBs. We showed that neither shear stress nor co-culture with pericytes could induce WPB formation. By further studying the metabolism with NMR we found that hiPSC-EC have a reduced glycolysis and lactate production and an increased intracellular pH (pHi). This coincides with a reduced expression of the proton coupled monocarboxylate transporter MCT1, which transports H+ and lactate into the cell to keep pHi in balance. Reducing the intracellular pH led to increased presence of functional VWF and maturation of WPB in hiPSC-ECs. In Chapter 4 we addressed the question how hiPSC-EC maintain their redoxbalance and produce enough ATP, since the vast majority of ATP and antioxidants of EC are obtained by glycolysis, which is significantly reduced in iPSC-ECs. We found that hiPSC-EC rely mainly on free fatty acid oxidation and presumably use NADPH do maintain their redox balance. This alternative metabolic state in hiPSCEC was found to be independent of the high expression of the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which regulates fatty acid storage, glucose metabolism, lipid uptake and mitochondrial biogenesis. Since PGC1α is directly activated by ROS and is the master regulator of the energy metabolism, the high levels of PGC1α expression in hiPSCECcould also be a consequence instead of the cause of the observed differences in metabolism. In Chapter 5, we studied the immunogenic surface of hiPSC-ECs, since transplantation induced rejection by the host immune system is an essential hurdle in usage of IPSC derived tissue. Previous studies suggested that IPSC derived cell recognition by the host immune system is diminished compared to transplantation of allogenic human alternatives. On the other hand, endothelial surface MHC class 1 and 2 molecules do play an active, ‘APC like’, role in adult immunity. Therefore, we characterized hiPSC-EC surface immune complexes, unstimulated and after cytokine stimulation. In addition we tested how the observed difference in expression could influence CD8 T-cell activation. Furthermore, we characterized the expression of complement inhibitors on the cell surface, necessary for the protection against unprovoked complement activation in the blood. Chapter 6 provides a summary and discussion of the observations in this thesis, including future perspectives on studying metabolism and metabolepigenetics in iPSC derived kidney organoids by mass spectrometry imaging.
- Published
- 2021
44. Structure modeling hints at a granular organization of the Golgi ribbon
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Karen M. Page, Jessica J. McCormack, Mafalda Lopes-da-Silva, Francesca Patella, Kimberly Harrison-Lavoie, Jemima J. Burden, Ying-Yi Bernadette Quah, Dominic Scaglioni, Francesco Ferraro, and Daniel F. Cutler
- Subjects
Weibel-Palade Bodies ,Physiology ,Endothelial Cells ,Golgi Apparatus ,Cell Biology ,Plant Science ,General Biochemistry, Genetics and Molecular Biology ,Exocytosis ,Structural Biology ,von Willebrand Factor ,General Agricultural and Biological Sciences ,Ecology, Evolution, Behavior and Systematics ,Cells, Cultured ,Developmental Biology ,Biotechnology - Abstract
Background In vertebrate cells, the Golgi functional subunits, mini-stacks, are linked into a tri-dimensional network. How this “ribbon” architecture relates to Golgi functions remains unclear. Are all connections between mini-stacks equal? Is the local structure of the ribbon of functional importance? These are difficult questions to address, without a quantifiable readout of the output of ribbon-embedded mini-stacks. Endothelial cells produce secretory granules, the Weibel-Palade bodies (WPB), whose von Willebrand Factor (VWF) cargo is central to hemostasis. The Golgi apparatus controls WPB size at both mini-stack and ribbon levels. Mini-stack dimensions delimit the size of VWF "boluses” whilst the ribbon architecture allows their linear co-packaging, thereby generating WPBs of different lengths. This Golgi/WPB size relationship suits mathematical analysis. Results WPB lengths were quantized as multiples of the bolus size and mathematical modeling simulated the effects of different Golgi ribbon organizations on WPB size, to be compared with the ground truth of experimental data. An initial simple model, with the Golgi as a single long ribbon composed of linearly interlinked mini-stacks, was refined to a collection of mini-ribbons and then to a mixture of mini-stack dimers plus long ribbon segments. Complementing these models with cell culture experiments led to novel findings. Firstly, one-bolus sized WPBs are secreted faster than larger secretory granules. Secondly, microtubule depolymerization unlinks the Golgi into equal proportions of mini-stack monomers and dimers. Kinetics of binding/unbinding of mini-stack monomers underpinning the presence of stable dimers was then simulated. Assuming that stable mini-stack dimers and monomers persist within the ribbon resulted in a final model that predicts a “breathing” arrangement of the Golgi, where monomer and dimer mini-stacks within longer structures undergo continuous linking/unlinking, consistent with experimentally observed WPB size distributions. Conclusions Hypothetical Golgi organizations were validated against a quantifiable secretory output. The best-fitting Golgi model, accounting for stable mini-stack dimers, is consistent with a highly dynamic ribbon structure, capable of rapid rearrangement. Our modeling exercise therefore predicts that at the fine-grained level the Golgi ribbon is more complex than generally thought. Future experiments will confirm whether such a ribbon organization is endothelial-specific or a general feature of vertebrate cells.
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- 2021
45. Arf GTPase-activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor
- Author
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Yuka Eura, Yoko Shiba, Yuji Shibasaki, Keigo Baba, Hikari Hataida, Naoya Inoue, Rika Kimura, Satoshi Waguri, Wei-Fen Ni, Koichi Kokame, Honoka Sasaki, Kuniaki Sasaki, Asano Watanabe, and Kosuke Kamon
- Subjects
ADP ribosylation factor ,QH301-705.5 ,Science ,arf ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Exocytosis ,symbols.namesake ,Downregulation and upregulation ,Von Willebrand factor ,GTP-Binding Proteins ,von Willebrand Factor ,Weibel–Palade body ,Humans ,Secretion ,Biology (General) ,Weibel-Palade Bodies ,Chemistry ,wpb ,GTPase-Activating Proteins ,Membrane Proteins ,Golgi apparatus ,Cell biology ,Protein Transport ,Secretory protein ,symbols ,biology.protein ,arfgap ,vwf ,secretory granule ,General Agricultural and Biological Sciences ,exocytosis ,Biogenesis ,Research Article - Abstract
Arf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors (Arfs), which are critical to form transport intermediates. ArfGAPs have been thought to be negative regulators of Arfs; however, accumulating evidence indicates that ArfGAPs are important for cargo sorting and promote membrane traffic. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPB biogenesis at the Golgi was reported to be regulated by Arf and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the role of ArfGAPs in the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. The results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. On the other hand, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA) or histamine stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected roles of ArfGAPs in vWF transport., Summary: The Arf GTPase-activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor.
- Published
- 2021
46. APOLD1 loss causes endothelial dysfunction involving cell junctions, cytoskeletal architecture, and Weibel-Palade bodies, while disrupting hemostasis.
- Author
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Stritt S, Nurden P, Nurden AT, Schved JF, Bordet JC, Roux M, Alessi MC, Trégouët DA, Mäkinen T, and Giansily-Blaizot M
- Subjects
- Humans, von Willebrand Factor genetics, Endothelial Cells physiology, Angiopoietin-2 genetics, Exocytosis physiology, Hemostasis, Intercellular Junctions, Weibel-Palade Bodies, Vascular Diseases
- Abstract
Vascular homeostasis is impaired in various diseases thereby contributing to the progression of their underlying pathologies. The endothelial immediate early gene Apolipoprotein L domain-containing 1 (APOLD1) helps to regulate endothelial function. However, its precise role in endothelial cell biology remains unclear. We have localized APOLD1 to endothelial cell contacts and to Weibel-Palade bodies (WPB) where it associates with von Willebrand factor (VWF) tubules. Silencing of APOLD1 in primary human endothelial cells disrupted the cell junction-cytoskeletal interface, thereby altering endothelial permeability accompanied by spontaneous release of WPB contents. This resulted in an increased presence of WPB cargoes, notably VWF and angiopoietin-2 in the extracellular medium. Autophagy flux, previously recognized as an essential mechanism for the regulated release of WPB, was impaired in the absence of APOLD1. In addition, we report APOLD1 as a candidate gene for a novel inherited bleeding disorder across three generations of a large family in which an atypical bleeding diathesis was associated with episodic impaired microcirculation. A dominant heterozygous nonsense APOLD1:p.R49* variant segregated to affected family members. Compromised vascular integrity resulting from an excess of plasma angiopoietin-2, and locally impaired availability of VWF may explain the unusual clinical profile of APOLD1:p.R49* patients. In summary, our findings identify APOLD1 as an important regulator of vascular homeostasis and raise the need to consider testing of endothelial cell function in patients with inherited bleeding disorders without apparent platelet or coagulation defects.
- Published
- 2023
- Full Text
- View/download PDF
47. Mutations in Neurobeachin-like 2 do not impact Weibel-Palade body biogenesis and von Willebrand factor secretion in gray platelet syndrome Endothelial Colony Forming Cells.
- Author
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Kat M, van Moort I, Bürgisser PE, Kuijpers TW, Hofman M, Favier M, Favier R, Margadant C, Voorberg J, and Bierings R
- Abstract
Background: Patients with gray platelet syndrome (GPS) and Neurobeachin-like 2 (NBEAL2) deficiency produce platelets lacking alpha-granules (AGs) and present with lifelong bleeding symptoms. AGs are lysosome-related organelles and store the hemostatic protein von Willebrand factor (VWF) and the transmembrane protein P-selectin. Weibel-Palade bodies (WPBs) are lysosome-related organelles of endothelial cells and also store VWF and P-selectin. In megakaryocytes, NBEAL2 links P-selectin on AGs to the SNARE protein SEC22B on the endoplasmic reticulum, thereby preventing premature release of cargo from AG precursors. In endothelial cells, SEC22B drives VWF trafficking from the endoplasmic reticulum to Golgi and promotes the formation of elongated WPBs, but it is unclear whether this requires NBEAL2., Objectives: To investigate a potential role for NBEAL2 in WPB biogenesis and VWF secretion using NBEAL2-deficient endothelial cells., Methods: The interaction of SEC22B with NBEAL2 in endothelial cells was investigated by interatomic mass spectrometry and pull-down analysis. Endothelial colony forming cells were isolated from healthy controls and 3 unrelated patients with GPS and mutations in NBEAL2 ., Results: We showed that SEC22B binds to NBEAL2 in ECs. Endothelial colony forming cells derived from a patient with GPS are deficient in NBEAL2 but reveal normal formation and maturation of WPBs and normal WPB cargo recruitment. Neither basal nor histamine-induced VWF secretion is altered in the absence of NBEAL2., Conclusions: Although NBEAL2 deficiency causes the absence of AGs in patients with GPS, it does not impact WPB functionality in ECs. Our data highlight the differences in the regulatory mechanisms between these 2 hemostatic storage compartments., (© 2023 The Author(s).)
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- 2023
- Full Text
- View/download PDF
48. Annexins A2 and A8 in endothelial cell exocytosis and the control of vascular homeostasis.
- Author
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Gerke, Volker
- Subjects
- *
ANNEXINS , *ENDOTHELIAL cells , *EXOCYTOSIS , *HOMEOSTASIS , *BLOOD vessels , *BLOOD cells - Abstract
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. [ABSTRACT FROM AUTHOR]
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- 2016
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- View/download PDF
49. IFN-α, IFN-β, and IFN-γ Have Different Effect on the Production of Proinflammatory Factors Deposited in Weibel-Palade Bodies of Endothelial Cells Infected with Herpes Simplex Virus Type 1.
- Author
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Shcheglovitova, O., Boldyreva, N., Sklyankina, N., Babayants, A., and Frolova, I.
- Subjects
- *
INTERFERONS , *ENDOTHELIAL cells , *HERPES simplex virus , *PREPROENDOTHELIN , *INTERLEUKIN-8 - Abstract
We demonstrated similarities and differences in the effects of IFN-α and IFN-β compared to IFN-γ on the production of factors deposited in the Weibel-Palade bodies in cultures of endothelial cells (intact and infected with herpes simplex virus 1). IFN-α and IFN-β reduced the content of von Willebrand factor, endothelin-1, and soluble P-selectin and increased IL-8 concentration in the culture medium of human umbilical vein endothelial cells. IFN-γ reduced the content of all studied factors in the endothelial cell culture medium. Possible mechanisms of these effects are discussed. [ABSTRACT FROM AUTHOR]
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- 2016
- Full Text
- View/download PDF
50. Intracellular targets: A multiple cargo transporting molecule
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Vassilios Moussis, Vassilios Tsikaris, Evgenia Fotou, Savvas Christoforidis, Sofia Zografou, Panos Kouklis, Christos G Papadopoulos, Alaxandra Ntoyhaniari, and Violetta Maltabe
- Subjects
Endosome ,Cell-Penetrating Peptides ,CDC42 ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Exocytosis ,Cell membrane ,Structural Biology ,von Willebrand Factor ,Drug Discovery ,medicine ,Small GTPase ,Molecular Biology ,Pharmacology ,Oligopeptide ,Weibel-Palade Bodies ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,General Medicine ,0104 chemical sciences ,medicine.anatomical_structure ,Membrane ,Biophysics ,Molecular Medicine ,Intracellular - Abstract
The generation of cell-penetrating peptides as cargo-delivery systems has produced an immense number of studies owing to the importance of these systems as tools to deliver molecules into the cells, as well as due to the interest to shed light into a yet unclear mechanism of the entrance of these peptides into the cells. However, many cell-penetrating peptides might present drawbacks due to causing cellular toxicity, or due to being entrapped in endosomes, or as a result of their degradation before they meet their target. In this work, a cargo transporting molecule, the Cell Penetrating Sequential Oligopeptide Carrier (CPSOC), formed by the repetitive -Lys-Aib-Cys- moiety, was tested for its ability to penetrate the cell membrane and transport the conjugated peptides into the cells. The cysteine residue anchors bioactive molecules through a stable thioether bond. The lysine supplies the positive charge to the construct, whereas the α-amino isobutyric acid is well known to induce helicoid conformation to the peptide backbone and protects from enzymatic degradation. The present study demonstrates that CPSOC penetrates the membrane transporting the conjugated cargo into the cell. When we tested CPSOC-conjugated peptides carrying critical domains of Cdc42, a small GTPase implicated in exocytosis, the internalized peptides were found to be functional because they inhibited exocytosis of von Willebrand factor from endothelial Weibel-Palade bodies a trafficking event depending on the Cdc42 protein. The data suggest that the carrier can deliver efficiently functional peptides into the cells, and thus, it can be used as a multiple-cargo transporting molecule.
- Published
- 2021
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