A CRISPR/Cas9 engineered Von Willebrand factor deficient endothelial cell model shows alternative trafficking of Weibel-Palade body proteins

ABSTRACT: 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 co-stored in WPBs are subject to alternative trafficking routes. In Von Willebrand disease (VWD) patients, the 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. To study the role of WPBs in endothelial cells using CRISPR-mediated knockout of VWF in BOECs. We used CRISPR/Cas9 gene 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. Two VWF-/- BOEC clones were obtained and were entirely devoid of WPBs, while overall cell morphology was unaltered. Several WPB proteins, including CD63, syntaxin-3 and the cargo proteins Ang-2, IL-6 and IL-8 showed alternative trafficking and secretion in absence of VWF. Interestingly, Ang-2 changed localization to the cell periphery and colocalized with Tie-2. 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.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Endothelial Cell

DISEASE(S): Disease Free

SUBMITTER: Ruben Bierings  

LAB HEAD: Dr.Ir. Ruben Bierings

PROVIDER: PXD013857 | Pride | 2019-11-11

REPOSITORIES: Pride