scholarly journals V-ATPase V0a1 promotes Weibel–Palade body biogenesis through the regulation of membrane fission

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Yasuo Yamazaki ◽  
Yuka Eura ◽  
Koichi Kokame
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Secretory Granules ◽  
Dominant Negative ◽  
Primary Role ◽  
Membrane Fission ◽  
Cellular Events ◽  
Molecular Events ◽  
Golgi Network ◽  
Von Willebrand

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.

scholarly journals Aftiphilin and γ-Synergin Are Required for Secretagogue Sensitivity of Weibel-Palade Bodies in Endothelial Cells

2008 ◽  
Vol 19 (12) ◽  
pp. 5072-5081 ◽  
Author(s):  
Winnie W.Y. Lui-Roberts ◽  
Francesco Ferraro ◽  
Thomas D. Nightingale ◽  
Daniel F. Cutler
Keyword(s):  
Endothelial Cells ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Adaptor Protein ◽  
Secretory Phenotype ◽  
Regulated Secretory Pathway ◽  
Golgi Network ◽  
Exocytic Pathway ◽  
Von Willebrand ◽  
Interfering Rna

Formation of secretory organelles requires the coupling of cargo selection to targeting into the correct exocytic pathway. Although the assembly of regulated secretory granules is driven in part by selective aggregation and retention of content, we recently reported that adaptor protein-1 (AP-1) recruitment of clathrin is essential to the initial formation of Weibel-Palade bodies (WPBs) at the trans-Golgi network. A selective co-aggregation process might include recruitment of components required for targeting to the regulated secretory pathway. However, we find that acquisition of the regulated secretory phenotype by WPBs in endothelial cells is coupled to but can be separated from formation of the distinctive granule core by ablation of the AP-1 effectors aftiphilin and γ-synergin. Their depletion by small interfering RNA leads to WPBs that fail to respond to secretagogue and release their content in an unregulated manner. We find that these non-responsive WPBs have density, markers of maturation, and highly multimerized von Willebrand factor similar to those of wild-type granules. Thus, by also recruiting aftiphilin/γ-synergin in addition to clathrin, AP-1 coordinates formation of WPBs with their acquisition of a regulated secretory phenotype.

VON WILLEBRAND FACTOR (vWF) PRO-POLYPEPTIDE IS REQUIRED FOR vWF MULTIMER FORMATION

1987 ◽  
Author(s):  
C L Verweij ◽  
M Hart ◽  
H Pannekoek
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Vascular Endothelial Cells ◽  
Expression System ◽  
Proteolytic Processing ◽  
Information Encoding ◽  
Interchain Interactions ◽  
Von Willebrand ◽  
Willebrand Factor

The von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized in vascular endothelial cells as a pre-pro-polypeptide with a highly repetitive domain structure, symbolized by the formula:(H)-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-(0H).A heterologous expression system, consisting of a monkey kidney cell line (C0S-1), transfected with full-length vWF cDNA, is shown to mimic the constitutively, secretory pathway of vWF in endothelial cells. The assembly of pro-vWF into multimers and the proteolytic processing of these structures is found to oro-ceed along the following, consecutive steps. Pro-vWF subunits associate to form dimers, a process that does not involve the pro-polypeptide of pro-vWF. This observation is derived from transfection of C0S-1 cells with vWF cDNA, lacking the genetic information encoding the pro-polypeptide, composed of the domains D1 and D2. Pro-vWF dimers are linked intracellularly to form a regular series of multimeric structures that are secreted and cannot be distinguished from those released constitutively by endothelial cells. The presence of the pro-polypeptide, embedded in pro-vWF, is obligatory for multimerization since the deletion mutant lacking the D1 and D2 domains fails to assemble beyond the dimer stage. It is argued that the D domains are involved in interchain interactions.

scholarly journals CD63 is a component of Weibel-Palade bodies of human endothelial cells

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1184-1191 ◽  
Author(s):  
UM Vischer ◽  
DD Wagner
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Western Blotting ◽  
Vascular Endothelial Cells ◽  
Secretory Granules ◽  
Positive Staining ◽  
Cell Fractionation ◽  
Glycosylation Pattern ◽  
Adhesion Proteins ◽  
Shape Distribution

Weibel-Palade bodies are secretory granules of vascular endothelial cells specialized in the storage of von Willebrand factor (vWF) and P- selectin, two adhesion proteins that can be rapidly mobilized to the cell surface by exocytosis in response to thrombin or other agonists. In this study, we attempted to identify additional components of Weibel- Palade bodies by raising monoclonal antibodies to these granules, purified by cell fractionation. One antibody, 2C6, was found to be specific for CD63, a membrane glycoprotein previously described in the lysosomes of platelets and other cell types. The immunopurified 2C6 antigen was recognized by an anti-CD63 reference antibody, 2.28, by Western blotting. Also, the biosynthetic profile of the 2C6 antigen in endothelial cells showed a nascent molecular mass and a glycosylation pattern identical to that of CD63. Immunofluorescence staining with 2C6 showed the lysosomes, and also elongated structures identified as Weibel-Palade bodies by their shape, distribution, and positive staining with anti-vWF antibodies, CD63 was also found by Western blotting of subcellular fractions highly enriched in Weibel-Palade bodies. Our results indicate that CD63 colocalizes with vWF and P- selectin in the Weibel-Palade bodies of endothelial cells, and together with these adhesion proteins it could be rapidly expressed on the cell surface in areas of vascular injury and inflammation.

scholarly journals Weibel-Palade body size modulates the adhesive activity of its von Willebrand Factor cargo in cultured endothelial cells

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Francesco Ferraro ◽  
Mafalda Lopes da Silva ◽  
William Grimes ◽  
Hwee Kuan Lee ◽  
Robin Ketteler ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Granules ◽  
Vascular Wall ◽  
Endothelial Cell Surface ◽  
Endothelial Surface ◽  
Wide Range ◽  
Storage Organelle ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Changes in the size of cellular organelles are often linked to modifications in their function. Endothelial cells store von Willebrand Factor (vWF), a glycoprotein essential to haemostasis in Weibel-Palade bodies (WPBs), cigar-shaped secretory granules that are generated in a wide range of sizes. We recently showed that forcing changes in the size of WPBs modifies the activity of this cargo. We now find that endothelial cells treated with statins produce shorter WPBs and that the vWF they release at exocytosis displays a reduced capability to recruit platelets to the endothelial cell surface. Investigating other functional consequences of size changes of WPBs, we also report that the endothelial surface-associated vWF formed at exocytosis recruits soluble plasma vWF and that this process is reduced by treatments that shorten WPBs, statins included. These results indicate that the post-exocytic adhesive activity of vWF towards platelets and plasma vWF at the endothelial surface reflects the size of their storage organelle. Our findings therefore show that changes in WPB size, by influencing the adhesive activity of its vWF cargo, may represent a novel mode of regulation of platelet aggregation at the vascular wall.

Small- and intermediate-conductance Ca2+-activated K+ channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells

2007 ◽  
Vol 293 (1) ◽  
pp. C458-C467 ◽  
Author(s):  
Jian-Zhong Sheng ◽  
Andrew P. Braun
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Early Event ◽  
No Production ◽  
Primary Role ◽  
Membrane Hyperpolarization ◽  
Intracellular Ca ◽  
Umbilical Vein Endothelial Cells

The contribution of small-conductance (SKCa) and intermediate-conductance Ca2+-activated K+ (IKCa) channels to the generation of nitric oxide (NO) by Ca2+-mobilizing stimuli was investigated in human umbilical vein endothelial cells (HUVECs) by combining single-cell microfluorimetry with perforated patch-clamp recordings to monitor agonist-evoked NO synthesis, cytosolic Ca2+ transients, and membrane hyperpolarization in real time. ATP or histamine evoked reproducible elevations in NO synthesis and cytosolic Ca2+, as judged by 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) and fluo-3 fluorescence, respectively, that were tightly associated with membrane hyperpolarizations. Whereas evoked NO synthesis was unaffected by either tetraethylammonium (10 mmol/l) or BaCl2 (50 μmol/l) + ouabain (100 μmol/l), depleting intracellular Ca2+ stores by thapsigargin or removing external Ca2+ inhibited NO production, as did exposure to high (80 mmol/l) external KCl. Importantly, apamin and charybdotoxin (ChTx)/ triarylmethane (TRAM)-34, selective blockers SKCa and IKCa channels, respectively, abolished both stimulated NO synthesis and membrane hyperpolarization and decreased evoked Ca2+ transients. Apamin and TRAM-34 also inhibited an agonist-induced outwardly rectifying current characteristic of SKCa and IKCa channels. Under voltage-clamp control, we further observed that the magnitude of agonist-induced NO production varied directly with the degree of membrane hyperpolarization. Mechanistically, our data indicate that SKCa and IKCa channel-mediated hyperpolarization represents a critical early event in agonist-evoked NO production by regulating the influx of Ca2+ responsible for endothelial NO synthase activation. Moreover, it appears that the primary role of agonist-induced release of intracellular Ca2+ stores is to trigger the opening of both KCa channels along with Ca2+ entry channels at the plasma membrane. Finally, the observed inhibition of stimulated NO synthesis by apamin and ChTx/TRAM-34 demonstrates that SKCa and IKCa channels are essential for NO-mediated vasorelaxation.

scholarly journals ETS transcription factor ETV2 directly converts human fibroblasts into functional endothelial cells

2014 ◽  
Vol 112 (1) ◽  
pp. 160-165 ◽  
Author(s):  
Rimpei Morita ◽  
Mayu Suzuki ◽  
Hidenori Kasahara ◽  
Nana Shimizu ◽  
Takashi Shichita ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factors ◽  
Vascular Endothelial Cells ◽  
Human Fibroblasts ◽  
Vascular Endothelial ◽  
Promising Therapeutic Approach ◽  
Minimum Number ◽  
Von Willebrand ◽  
Endothelial Development

Transplantation of endothelial cells (ECs) is a promising therapeutic approach for ischemic disorders. In addition, the generation of ECs has become increasingly important for providing vascular plexus to regenerated organs, such as the liver. Although many attempts have been made to generate ECs from pluripotent stem cells and nonvascular cells, the minimum number of transcription factors that specialize in directly inducing vascular ECs remains undefined. Here, by screening 18 transcription factors that are important for both endothelial and hematopoietic development, we demonstrate that ets variant 2 (ETV2) alone directly converts primary human adult skin fibroblasts into functional vascular endothelial cells (ETVECs). In coordination with endogenous FOXC2 in fibroblasts, transduced ETV2 elicits expression of multiple key endothelial development factors, including FLI1, ERG, and TAL1, and induces expression of endothelial functional molecules, including EGFL7 and von Willebrand factor. Consequently, ETVECs exhibits EC characteristics in vitro and forms mature functional vasculature in Matrigel plugs transplanted in NOD SCID mice. Furthermore, ETVECs significantly improve blood flow recovery in a hind limb ischemic model using BALB/c-nu mice. Our study indicates that the creation of ETVECs provides further understanding of human EC development induced by ETV2.

Perturbation of cultured human vascular endothelial cells by phorbol ester or thrombin alters the cellular von Willebrand factor distribution

1987 ◽  
Vol 133 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Jan Hendrik Reinders ◽  
Richard C. Vervoorn ◽  
Cornelis L. Verweij ◽  
Jan A. Van Mourik ◽  
Philip G. De Groot
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Phorbol Ester ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals The roles of protein kinase C and intracellular Ca2+ in the secretion of von Willebrand factor from human vascular endothelial cells

1992 ◽  
Vol 286 (2) ◽  
pp. 631-635 ◽  
Author(s):  
M A Carew ◽  
E M Paleolog ◽  
J D Pearson
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Selective Inhibition ◽  
Von Willebrand ◽  
Willebrand Factor

Secretion of von Willebrand factor (vWf) glycoprotein from storage granules in human umbilical-vein endothelial cells was studied in vitro. Either elevation of intracellular Ca2+ concentration ([Ca2+]i) with a Ca2+ ionophore or activation of protein kinase (PK) C by phorbol 12-myristate 13-acetate caused vWf secretion, and together the agents acted synergistically. However, when vWf release was stimulated by receptor-mediated agonists, selective inhibition of PKC had no effect on histamine-induced secretion and significantly elevated thrombin-induced secretion. Furthermore, ATP, which efficiently elevates [Ca2+]i in these cells, was a very poor effector of vWf release. We conclude that elevation of [Ca2+]i by physiological agonists is necessary for vWf release, but other signalling mechanisms, as yet uncharacterized, but not due to PKC activation, are required for full induction of the secretory pathway.

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