Dataset Information

Alternative trafficking of Weibel-Palade body proteins in CRISPR/Cas9-engineered von Willebrand factor-deficient blood outgrowth endothelial cells.

ABSTRACT: Background:Synthesis of the hemostatic protein von Willebrand factor (VWF) drives formation of endothelial storage organelles called Weibel-Palade bodies (WPBs). In the absence of VWF, angiogenic and inflammatory mediators that are costored in WPBs are subject to alternative trafficking routes. In patients with von Willebrand disease (VWD), partial or complete absence of VWF/WPBs may lead to additional bleeding complications, such as angiodysplasia. Studies addressing the role of VWF using VWD patient-derived blood outgrowth endothelial cells (BOECs) have reported conflicting results due to the intrinsic heterogeneity of patient-derived BOECs. Objective:To generate a VWF-deficient endothelial cell model using clustered regularly interspaced short palindromic repeats (CRISPR) genome engineering of blood outgrowth endothelial cells. Methods:We used CRISPR/CRISPR-associated protein 9 editing in single-donor cord blood-derived BOECs (cbBOECs) to generate clonal VWF -/- cbBOECs. Clones were selected using high-throughput screening, VWF mutations were validated by sequencing, and cells were phenotypically characterized. Results:Two VWF -/- BOEC clones were obtained and were entirely devoid of WPBs, while their overall cell morphology was unaltered. Several WPB proteins, including CD63, syntaxin-3 and the cargo proteins angiopoietin (Ang)-2, interleukin (IL)-6, and IL-8 showed alternative trafficking and secretion in the absence of VWF. Interestingly, Ang-2 was relocated to the cell periphery and colocalized with Tie-2. Conclusions:CRISPR editing of VWF provides a robust method to create VWF- deficient BOECs that can be directly compared to their wild-type counterparts. Results obtained with our model system confirmed alternative trafficking of several WPB proteins in the absence of VWF and support the theory that increased Ang-2/Tie-2 interaction contributes to angiogenic abnormalities in VWD patients.

SUBMITTER: Schillemans M

PROVIDER: S-EPMC6782018 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Alternative trafficking of Weibel-Palade body proteins in CRISPR/Cas9-engineered von Willebrand factor-deficient blood outgrowth endothelial cells.

Schillemans Maaike M Kat Marije M Westeneng Jurjen J Gangaev Anastasia A Hofman Menno M Nota Benjamin B van Alphen Floris P J FPJ de Boer Martin M van den Biggelaar Maartje M Margadant Coert C Voorberg Jan J Bierings Ruben R

Research and practice in thrombosis and haemostasis 20190801 4

<h4>Background</h4>Synthesis of the hemostatic protein von Willebrand factor (VWF) drives formation of endothelial storage organelles called Weibel-Palade bodies (WPBs). In the absence of VWF, angiogenic and inflammatory mediators that are costored in WPBs are subject to alternative trafficking routes. In patients with von Willebrand disease (VWD), partial or complete absence of VWF/WPBs may lead to additional bleeding complications, such as angiodysplasia. Studies addressing the role of VWF usi ...[more]

PMID: 31624792

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Project description:BackgroundWeibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). The temperature-dependence of Ca(2+)-driven WPB exocytosis is not known, although indirect evidence suggests that WPB exocytosis may occur at very low temperatures. Here we quantitatively analyse the temperature-dependence of Ca(2+)-driven WPB exocytosis and release of secreted VWF from the cell surface of ECs using fluorescence microscopy of cultured human ECs containing fluorescent WPBs.Principal findingsCa(2+)-driven WPB exocytosis occurred at all temperatures studied (7-37°C). The kinetics and extent of WPB exocytosis were strongly temperature-dependent: Delays in exocytosis increased from 0.92 s at 37°C to 134.2 s at 7°C, the maximum rate of WPB fusion decreased from 10.0±2.2 s(-1) (37°C) to 0.80±0.14 s(-1) (7°C) and the fractional extent of degranulation of WPBs in each cell from 67±3% (37°C) to 3.6±1.3% (7°C). A discrepancy was found between the reduction in Ca(2+)-driven VWF secretion and WPB exocytosis at reduced temperature; at 17°C VWF secretion was reduced by 95% but WPB exocytosis by 75-80%. This discrepancy arises because VWF dispersal from sites of WPB exocytosis is largely prevented at low temperature. In contrast VWF-propolypeptide (proregion) dispersal from WPBs, although slowed, was complete within 60-120 s. Novel antibodies to the cleaved and processed proregion were characterised and used to show that secreted proregion more accurately reports the secretion of WPBs at sub-physiological temperatures than assay of VWF itself.ConclusionsWe report the first quantitative analysis of the temperature-dependence of WPB exocytosis. We provide evidence; by comparison of biochemical data for VWF or proregion secretion with direct analysis of WPB exocytosis at reduced temperature, that proregion is a more reliable marker for WPB exocytosis at reduced temperature, where VWF-EC adhesion is increased.

Dataset Information

Alternative trafficking of Weibel-Palade body proteins in CRISPR/Cas9-engineered von Willebrand factor-deficient blood outgrowth endothelial cells.

Publications

Alternative trafficking of Weibel-Palade body proteins in CRISPR/Cas9-engineered von Willebrand factor-deficient blood outgrowth endothelial cells.

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