Open AccessAlternative trafficking of Weibel‐Palade body proteins in CRISPR /Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cells
Author(s) -
Schillemans Maaike,
Kat Marije,
Westeneng Jurjen,
Gangaev Anastasia,
Hofman Menno,
Nota Benjamin,
van Alphen Floris P. J.,
de Boer Martin,
van den Biggelaar Maartje,
Margadant Coert,
Voorberg Jan,
Bierings Ruben
Publication year - 2019
Publication title -
research and practice in thrombosis and haemostasis
Language(s) - English
Resource type - Journals
ISSN - 2475-0379
DOI - 10.1002/rth2.12242
Subject(s) - von willebrand factor , crispr , von willebrand disease , biology , genome editing , microbiology and biotechnology , immunology , chemistry , gene , genetics , platelet
Background Synthesis of the hemostatic protein von Willebrand factor ( VWF ) drives formation of endothelial storage organelles called Weibel‐Palade bodies ( WPB s). In the absence of VWF , angiogenic and inflammatory mediators that are costored in WPB s are subject to alternative trafficking routes. In patients with von Willebrand disease ( VWD ), partial or complete absence of VWF / WPB s may lead to additional bleeding complications, such as angiodysplasia. Studies addressing the role of VWF using VWD patient–derived blood outgrowth endothelial cells ( BOEC s) have reported conflicting results due to the intrinsic heterogeneity of patient‐derived BOEC s. 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 BOEC s (cb BOEC s) to generate clonal VWF −/− cb BOEC s. 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 WPB s, while their overall cell morphology was unaltered. Several WPB proteins, including CD 63, 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 BOEC s 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.