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We studied pedestrians' perception of their ability to see when outside after dark, the luminance of the pavement after dark and the association between perception and luminance. These data were captured by a citizen science network of smartphone users, with and without an eye disease. They used an app to report their ability to see when outside after dark in their own neighbourhood and measured the luminance of the pavement using the smartphone camera. Logistic regression was used to determine the influence of luminance, age, gender and eye disease on reported ability to see after dark. Amongst those respondents who did not report an eye disease, 11% reported visual conditions they perceived to make walking difficult; this increased to 40% for pedestrians who reported an eye disease. The recorded luminances were typically 0.01–0.1 cd/m2. For those respondents with healthy eyes, the percentage reporting difficult visual conditions increased especially below 0.01 cd/m2; for those with an eye disease, the increase started at higher luminances, which may limit their mobility after dark. [ABSTRACT FROM AUTHOR]

Caenorhabditis elegans CEP-1 activates germline apoptosis in response to genotoxic stress, similar to its mammalian counterpart, tumor suppressor p53. In mammals, there are three p53 family members (p53, p63, and p73) that activate and repress many distinct and overlapping sets of genes, revealing a complex transcriptional regulatory network. Because CEP-1 is the sole p53 family member in C. elegans, analysis of this network is greatly simplified in this organism. We found that CEP-1 functions during normal development in the absence of stress to repress many (331) genes and activate only a few (28) genes. In response to genotoxic stress, 1394 genes are activated and 942 are repressed, many of which contain p53-binding sites. Comparison of the CEP-1 transcriptional network with transcriptional targets of the human p53 family reveals considerable overlap between CEP-1-regulated genes and homologues regulated by human p63 and p53, suggesting a composite p53/p63 action for CEP-1. We found that phg-1, the C. elegans Gas1 (growth arrest-specific 1) homologue, is activated by CEP-1 and is a negative regulator of cell proliferation in the germline in response to genotoxic stress. Further, we find that CEP-1 and PHG-1 mediate the decreased developmental rate and embryonic viability of mutations in the clk-2/TEL2 gene, which regulates lifespan and checkpoint responses.Cell Death and Differentiation (2007) 14, 662–670. doi:10.1038/sj.cdd.4402075; published online 22 December 2006 [ABSTRACT FROM AUTHOR]

Background: Von Willebrand disease (VWD) is the most common inherited bleeding disorder caused by quantitative or qualitative defects of VWF. The p.M771V VWF variant leads to a severe bleeding phenotype in homozygous patients. However, the exact molecular mechanism remains unclear, which prevents personalized treatment of those VWD patients., Objective: This study aims to characterize the underlying molecular mechanisms of the p.M771V variant in multiple representative ex-vivo cell models., Methods: ECFCs were isolated from venous blood of VWD patients from the Willebrand in the Netherlands (WiN) cohort carrying homozygous and heterozygous p.M771V VWF variants. The p.M771V variant was also introduced in cord-blood derived ECFCs (CB-ECFCs) through adenine base editing and was overexpressed in HEK293 cells. Biosynthesis, storage, and secretion of VWF was studied using biochemical methods and confocal microscopy., Results: Two unrelated homozygous p.M771V patients presented with very low VWF activity and antigen levels in plasma. Patient ECFCs showed impaired proVWF processing into mature VWF with secreted VWF being severely reduced when compared to ECFCs of healthy donors. Multimer analysis of p.M771V ECFCs showed a deficiency of high molecular weight VWF multimers. Immunofluorescent staining revealed VWF retention in the endoplasmic reticulum (ER); this was confirmed in various populations of base edited CB-ECFCs harboring the p.M771V variant., Conclusion: The severe endothelial phenotype observed in patient-derived p.M771V ECFCs, HEK293 cells, and an original base-edited CB-ECFC modelling system, show that ER retention of VWF and failure to undergo subsequent proteolytic processing underpins the severe bleeding phenotype of patients with homozygous variants at M771., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.)

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.)

Background: von Willebrand disease (VWD) is the most common inherited bleeding disorder, characterized by either partial or complete von Willebrand factor (VWF) deficiency or by the occurrence of VWF proteoforms of altered functionality. The gene encoding VWF is highly polymorphic, giving rise to a variety of proteoforms with varying plasma concentrations and clinical significance., Objectives: To address this complexity, we translated genomic variation in VWF to corresponding VWF proteoforms circulating in blood., Methods: VWF was characterized in VWD patients (n = 64) participating in the Willebrand in the Netherlands study by conventional laboratory testing, DNA sequencing and complementary discovery, and targeted mass spectrometry-based plasma proteomic strategies., Results: Unbiased plasma profiling combined with immune enrichment of VWF verified VWF and its binding partner factor VIII as key determinants of VWD and revealed a remarkable heterogeneity in VWF amino acid sequence coverage among patients. Subsequent VWF proteotyping enabled identification of both polymorphisms (eg, p.Thr789Ala, p.Gln852Arg, and p.Thr1381Ala), as well as pathogenic variants (n = 16) along with their corresponding canonical sequences. Targeted proteomics using stable isotope-labeled peptides confirmed unbiased proteotyping for 5 selected variants and suggested differential proteoform quantities in plasma. The variant-to-wild-type peptide ratio was determined in 6 type 2B patients heterozygous for p.Arg1306Trp, confirming the relatively low proteoform concentration of the pathogenic variant. The elevated VWF propeptide/VWF ratio indicated increased clearance of specific VWF proteoforms., Conclusion: This study highlights how VWF proteotyping from plasma could be the first step to bridge the gap between genotyping and functional testing in VWD., 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.)

Abstract: von Willebrand factor (VWF) undergoes complex posttranslational modification within endothelial cells (ECs) before secretion. This includes significant N- and O-linked glycosylation. Previous studies have demonstrated that changes in N-linked glycan structures significantly influence VWF biosynthesis. In contrast, although abnormalities in VWF O-linked glycans (OLGs) have been associated with enhanced VWF clearance, their effect on VWF biosynthesis remains poorly explored. Herein, we report a novel role for OLG determinants in regulating VWF biosynthesis and trafficking within ECs. We demonstrate that alterations in OLGs (notably reduced terminal sialylation) lead to activation of the A1 domain of VWF within EC. In the presence of altered OLG, VWF multimerization is reduced and Weibel-Palade body (WPB) formation significantly impaired. Consistently, the amount of VWF secreted from WPB after EC activation was significantly reduced in the context of O-glycosylation inhibition. Finally, altered OLG on VWF not only reduced the amount of VWF secreted after EC activation but also affected its hemostatic efficacy. Notably, VWF secreted after WPB exocytosis consisted predominantly of low molecular weight multimers, and the length of tethered VWF string formation on the surface of activated ECs was significantly reduced. In conclusion, our data therefore support the hypothesis that alterations in O-glycosylation pathways directly affect VWF trafficking within human EC. These findings are interesting given that previous studies have reported altered OLG on plasma VWF (notably increased T-antigen expression) in patients with von Willebrand disease., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

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.)

Background: Von Willebrand factor (VWF) and VWF propeptide (VWFpp) are stored in eccentric nanodomains within platelet alpha-granules. VWF and VWFpp can undergo differential secretion following Weibel-Palade body exocytosis in endothelial cells; however, it is unclear if the same process occurs during platelet alpha-granule exocytosis. Using a high-throughput 3-dimensional super-resolution imaging workflow for quantification of individual platelet alpha-granule cargo, we studied alpha-granule cargo release in response to different physiological stimuli., Objectives: To investigate how VWF and VWFpp are released from alpha-granules in response to physiological stimuli., Methods: Platelets were activated with protease-activated receptor 1 (PAR-1) activating peptide (PAR-1 ap) or collagen-related peptide (CRP-XL). Alpha-tubulin, VWF, VWFpp, secreted protein acidic and cysteine rich (SPARC), and fibrinogen were imaged using 3-dimensional structured illumination microscopy, followed by semiautomated analysis in FIJI. Uptake of anti-VWF nanobody during degranulation was used to identify alpha-granules that partially released content., Results: VWFpp overlapped with VWF in eccentric alpha-granule subdomains in resting platelets and showed a higher degree of overlap with VWF than SPARC or fibrinogen. Activation of PAR-1 (0.6-20 μM PAR-1 ap) or glycoprotein VI (GPVI) (0.25-1 μg/mL CRP-XL) signaling pathways caused a dose-dependent increase in alpha-granule exocytosis. More than 80% of alpha-granules remained positive for VWF, even at the highest agonist concentrations. In contrast, the residual fraction of alpha-granules containing VWFpp decreased in a dose-dependent manner to 23%, whereas SPARC and fibrinogen were detected in 60% to 70% of alpha-granules when stimulated with 20 μM PAR-1 ap. Similar results were obtained using CRP-XL. Using an extracellular anti-VWF nanobody, we identified VWF in postexocytotic alpha-granules., Conclusion: We provide evidence for differential secretion of VWF and VWFpp from individual alpha-granules., Competing Interests: Declaration of competing interests F.W.G.L. received research support from CSL Behring, Takeda, uniQure, and Sobi and is a consultant for uniQure, Biomarin, CSL Behring, and Takeda, of which the fees go to the institute. He was a DSMB member for a study sponsored by Roche. A.J.G.J. received speaker fees and travel cost payments from 3SBio, Amgen, and Novartis, is on the international advisory board at Novartis, and received research support from CSL Behring, Principia, and Argenx. None of the other authors have conflicts of interest to declare., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

One of the most used and versatile methods to study number, dimensions, content and localization of secretory organelles is confocal microscopy analysis. However, considerable heterogeneity exists in the number, size and shape of secretory organelles that can be present in the cell. One thus needs to analyze large numbers of organelles for valid quantification. Properly evaluating these parameters requires an automated, unbiased method to process and quantitatively analyze microscopy data. Here, we describe two pipelines, run by CellProfiler software, called OrganelleProfiler and OrganelleContentProfiler. These pipelines were used on confocal images of endothelial colony forming cells (ECFCs), which contain unique secretory organelles called Weibel-Palade bodies (WPBs), and on early endosomes in ECFCs and human embryonic kidney 293T (HEK293T) cells. Results show that the pipelines can quantify the cell count, size, organelle count, organelle size, shape, relation to cells and nuclei, and distance to these objects in both endothelial and HEK293T cells. Additionally, the pipelines were used to measure the reduction in WPB size after disruption of the Golgi and to quantify the perinuclear clustering of WPBs after triggering of cAMP-mediated signaling pathways in ECFCs. Furthermore, the pipeline is able to quantify secondary signals located in or on the organelle or in the cytoplasm, such as the small WPB GTPase Rab27A. Cell profiler measurements were checked for validity using Fiji. To conclude, these pipelines provide a powerful, high-processing quantitative tool for the characterization of multiple cell and organelle types. These pipelines are freely available and easily editable for use on different cell types or organelles., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Laan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The assembly of von Willebrand factor (VWF) into ordered helical tubules within endothelial Weibel-Palade bodies (WPBs) is required for the efficient deployment of the protein at sites of vascular injury. VWF trafficking and storage are sensitive to cellular and environmental stresses that are associated with heart disease and heart failure. Altered storage of VWF manifests as a change in WPB morphology from a rod shape to a rounded shape and is associated with impaired VWF deployment during secretion. In this study, we examined the morphology, ultrastructure, molecular composition and kinetics of exocytosis of WPBs in cardiac microvascular endothelial cells isolated from explanted hearts of patients with a common form of heart failure, dilated cardiomyopathy (DCM; HCMEC D ), or from nominally healthy donors (controls; HCMEC C ). Using fluorescence microscopy, WPBs in HCMEC C (n = 3 donors) showed the typical rod-shaped morphology containing VWF, P-selectin and tPA. In contrast, WPBs in primary cultures of HCMEC D (n = 6 donors) were predominantly rounded in shape and lacked tissue plasminogen activator (t-PA). Ultrastructural analysis of HCMEC D revealed a disordered arrangement of VWF tubules in nascent WPBs emerging from the trans-Golgi network. HCMEC D WPBs still recruited Rab27A, Rab3B, Myosin-Rab Interacting Protein (MyRIP) and Synaptotagmin-like protein 4a (Slp4-a) and underwent regulated exocytosis with kinetics similar to that seen in HCMECc. However, secreted extracellular VWF strings from HCMEC D were significantly shorter than for endothelial cells with rod-shaped WPBs, although VWF platelet binding was similar. Our observations suggest that VWF trafficking, storage and haemostatic potential are perturbed in HCMEC from DCM hearts.

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).)