Genetically Engineered Endothelial Models Uncover Dysregulated Trafficking of Angiopoietin-2 and Nuclear Reorganization in VWF Deficiency

Introduction Von Willebrand factor (VWF) is essential for Weibel-Palade body (WPB) formation and regulated storage of inflammatory proteins in endothelial cells. Mutations affecting VWF biosynthesis may disrupt intracellular trafficking and secretion pathways. Here, we modeled a large VWF deletion (exons 4–34) previously identified in a type 3 VWD patient, to investigate its impact on endothelial organelle integrity and angiopoietin-2 (Ang2) trafficking. Method An innovative CRISPR/Cas9 approach was employed using four guide RNAs targeting flanking regions of exons 4–34 to induce a ~100 kb deletion in healthy endothelial colony-forming cells (ECFCs) and human umbilical vein endothelial cells (HUVECs). In HUVECs, single-cell colonies were established for detailed phenotypic and colocalization analysis. For transcriptomic analysis, RNA sequencing (RNA-seq) was performed on bulk CRISPR-edited ECFCs. Total RNA was processed using the QuantSeq 3′ mRNA Library Prep Kit, and sequencing was carried out on an Illumina NovaSeq 6000 platform. Gene Ontology – Cellular Component enrichment analysis was performed on differentially expressed genes. Immunostaining imaging was performed to assess WPB morphology and the localization of Ang2. Z-stack images were acquired using a ZEISS ApoTome.2 microscope, and image analysis was conducted using ZEN Blue Edition software (ZEISS). To assess subcellular colocalization of Ang2 with organelle markers (histone H3, lamin B, NUP153, LC3AB, calnexin, TGN46), quantitative analysis was performed using Pearson’s correlation and Manders’ overlap coefficients. Results Both ECFCs and HUVECs with the VWF deletion showed absence or malformation of WPBs and altered intracellular trafficking of Ang2. Mislocalization of Ang2 toward the nucleus and perinuclear region was observed, supported by higher colocalization values with histone H3, lamin B, and NUP153. In parallel, reduced colocalization with the autophagy marker LC3AB suggested impaired vesicular storage and loss of WPB-like structures. Preliminary RNA-seq and GO component analysis revealed transcriptomic shifts affecting compartments such as secretory organelles, ER-Golgi intermediate compartment membrane, recycling endosome membrane, lysosomal lumen, perinuclear region of cytoplasm, extracellular exosomes, and nucleoplasm—partially consistent with the altered Ang2 localization observed by imaging. Conclusion CRISPR/Cas9-induced deletion of VWF in ECFCs and HUVECs successfully recapitulates WPB abnormalities observed in patient-derived cells and highlights a profound shift in Ang2 trafficking toward nuclear and perinuclear compartments. Combined imaging and transcriptomic data indicate that loss of VWF disrupts WPB formation and broader intracellular pathways linked to angiogenesis and inflammation. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany