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 rou...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2019-10-01
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| Series: | Research and Practice in Thrombosis and Haemostasis |
| Subjects: | |
| Online Access: | https://doi.org/10.1002/rth2.12242 |
| _version_ | 1827845652169621504 |
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| author | Maaike Schillemans Marije Kat Jurjen Westeneng Anastasia Gangaev Menno Hofman Benjamin Nota Floris P. J. van Alphen Martin de Boer Maartje van den Biggelaar Coert Margadant Jan Voorberg Ruben Bierings |
| author_facet | Maaike Schillemans Marije Kat Jurjen Westeneng Anastasia Gangaev Menno Hofman Benjamin Nota Floris P. J. van Alphen Martin de Boer Maartje van den Biggelaar Coert Margadant Jan Voorberg Ruben Bierings |
| author_sort | Maaike Schillemans |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-12T09:00:16Z |
| format | Article |
| id | doaj.art-f5bad20c9eee478db4af6502d1df88c3 |
| institution | Directory Open Access Journal |
| issn | 2475-0379 |
| language | English |
| last_indexed | 2024-03-12T09:00:16Z |
| publishDate | 2019-10-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Research and Practice in Thrombosis and Haemostasis |
| spelling | doaj.art-f5bad20c9eee478db4af6502d1df88c32023-09-02T15:44:52ZengElsevierResearch and Practice in Thrombosis and Haemostasis2475-03792019-10-013471873210.1002/rth2.12242Alternative trafficking of Weibel‐Palade body proteins in CRISPR/Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cellsMaaike Schillemans0Marije Kat1Jurjen Westeneng2Anastasia Gangaev3Menno Hofman4Benjamin Nota5Floris P. J. van Alphen6Martin de Boer7Maartje van den Biggelaar8Coert Margadant9Jan Voorberg10Ruben Bierings11Molecular and Cellular Hemostasis Sanquin Research and Landsteiner Laboratory Amsterdam UMCUniversity of Amsterdam Amsterdam The NetherlandsMolecular and Cellular Hemostasis Sanquin Research and Landsteiner Laboratory Amsterdam UMCUniversity of Amsterdam Amsterdam The NetherlandsMolecular and Cellular Hemostasis Sanquin Research and Landsteiner Laboratory Amsterdam UMCUniversity of Amsterdam Amsterdam The NetherlandsMolecular and Cellular Hemostasis Sanquin Research and Landsteiner Laboratory Amsterdam UMCUniversity of Amsterdam Amsterdam The NetherlandsMolecular and Cellular Hemostasis Sanquin Research and Landsteiner Laboratory Amsterdam UMCUniversity of Amsterdam Amsterdam The NetherlandsMolecular and Cellular Hemostasis Sanquin Research and Landsteiner Laboratory Amsterdam UMCUniversity of Amsterdam Amsterdam The NetherlandsMolecular and Cellular Hemostasis Sanquin Research and Landsteiner Laboratory Amsterdam UMCUniversity of Amsterdam Amsterdam The NetherlandsBlood Cell Research Sanquin Research and Landsteiner Laboratory Amsterdam UMCUniversity of Amsterdam Amsterdam The NetherlandsMolecular and Cellular Hemostasis Sanquin Research and Landsteiner Laboratory Amsterdam UMCUniversity of Amsterdam Amsterdam The NetherlandsMolecular and Cellular Hemostasis Sanquin Research and Landsteiner Laboratory Amsterdam UMCUniversity of Amsterdam Amsterdam The NetherlandsMolecular and Cellular Hemostasis Sanquin Research and Landsteiner Laboratory Amsterdam UMCUniversity of Amsterdam Amsterdam The NetherlandsMolecular and Cellular Hemostasis Sanquin Research and Landsteiner Laboratory Amsterdam UMCUniversity of Amsterdam Amsterdam The NetherlandsAbstract 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.https://doi.org/10.1002/rth2.12242endothelial cellsgene knockout techniquesprotein transportsecretory vesiclesvon Willebrand factor |
| spellingShingle | Maaike Schillemans Marije Kat Jurjen Westeneng Anastasia Gangaev Menno Hofman Benjamin Nota Floris P. J. van Alphen Martin de Boer Maartje van den Biggelaar Coert Margadant Jan Voorberg Ruben Bierings Alternative trafficking of Weibel‐Palade body proteins in CRISPR/Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cells Research and Practice in Thrombosis and Haemostasis endothelial cells gene knockout techniques protein transport secretory vesicles von Willebrand factor |
| title | Alternative trafficking of Weibel‐Palade body proteins in CRISPR/Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cells |
| title_full | Alternative trafficking of Weibel‐Palade body proteins in CRISPR/Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cells |
| title_fullStr | Alternative trafficking of Weibel‐Palade body proteins in CRISPR/Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cells |
| title_full_unstemmed | Alternative trafficking of Weibel‐Palade body proteins in CRISPR/Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cells |
| title_short | Alternative trafficking of Weibel‐Palade body proteins in CRISPR/Cas9‐engineered von Willebrand factor–deficient blood outgrowth endothelial cells |
| title_sort | alternative trafficking of weibel palade body proteins in crispr cas9 engineered von willebrand factor deficient blood outgrowth endothelial cells |
| topic | endothelial cells gene knockout techniques protein transport secretory vesicles von Willebrand factor |
| url | https://doi.org/10.1002/rth2.12242 |
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