Dataset Information

Von Willebrand factor propeptide variants lead to impaired storage and ER retention in patient-derived endothelial colony-forming cells.

ABSTRACT:

Background

von Willebrand factor (VWF) is synthesized by vascular endothelial cells and megakaryocytes. The VWF propeptide is critical for multimerization and acts as an intra-molecular chaperone for mature VWF in sorting to its storage organelles, Weibel-Palade bodies (WPBs). In the Canadian Type 3 VWD study, almost half of the identified variants were in the VWF propeptide and these were associated with an increased bleeding phenotype.

Objective

To investigate VWF propeptide variants that cause quantitative von Willebrand disease (VWD) by utilizing patient-derived endothelial colony-forming cells (ECFCs).

Patients/methods

Endothelial colony-forming cells were isolated from five Type 3 VWD patients from four families with the following variants: (1) homozygous p.Asp75_Gly178del (deletion of exons 4 and 5 deletion; Ex4-5del), (2) homozygous p.Cys633Arg, (3) homozygous p.Arg273Trp, and (4) p.Pro293Glnfs*164 and p.Gln419* inherited in the compound heterozygous state. Additionally, ECFCs were isolated from six family members (two Type 1 VWD, four unaffected).

Results

Endothelial colony-forming cells from the Type 3 patient with the compound heterozygous genotype exhibited a true null VWF cellular phenotype, with negligible VWF detected. In contrast, the other three propeptide variants presented a similar expression pattern in homozygous ECFCs where VWF was synthesized but not packaged in WPBs, and variant VWF had an increased association with the endoplasmic reticulum (ER) marker, protein disulfide-isomerase (PDI), indicating an ER-retention phenotype. The biosynthetic phenotype was similar but to a lesser degree in heterozygous ECFCs expressing the non-null variants.

Conclusion

This study further elucidates the importance of the VWF propeptide in the VWD phenotype using patient-derived cells.

SUBMITTER: Bowman M

PROVIDER: S-EPMC9246936 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Publications

von Willebrand factor propeptide variants lead to impaired storage and ER retention in patient-derived endothelial colony-forming cells.

Bowman Mackenzie M Casey Lara L Selvam Soundarya N SN Lima Patricia D A PDA Rawley Orla O Hinds Megan M Tuttle Angie A Grabell Julie J Iorio Alfonso A Walker Irwin I Lillicrap David D James Paula P

Journal of thrombosis and haemostasis : JTH 20220503 7

<h4>Background</h4>von Willebrand factor (VWF) is synthesized by vascular endothelial cells and megakaryocytes. The VWF propeptide is critical for multimerization and acts as an intra-molecular chaperone for mature VWF in sorting to its storage organelles, Weibel-Palade bodies (WPBs). In the Canadian Type 3 VWD study, almost half of the identified variants were in the VWF propeptide and these were associated with an increased bleeding phenotype.<h4>Objective</h4>To investigate VWF propeptide var ...[more]

PMID: 35466528

Similar Datasets

Project description:Essentials Endothelial colony forming cells (ECFCs) are a powerful tool to study vascular diseases ex vivo. Separate ECFC lines show variations in morphology and von Willebrand factor-related parameters. Maximum cell density is correlated with von Willebrand factor expression in ECFCs. Variations in ECFC lines are dependent on the age and mesenchymal state of the cells. ABSTRACT: Background Endothelial colony forming cells (ECFCs) are cultured endothelial cells derived from peripheral blood. ECFCs are a powerful tool to study pathophysiological mechanisms underlying vascular diseases, including von Willebrand disease. In prior research, however, large variations between ECFC lines were observed in, among others, von Willebrand factor (VWF) expression. Objective Understand the relation between phenotypic characteristics and VWF-related parameters of healthy control ECFCs. Methods ECFC lines (n = 16) derived from six donors were studied at maximum cell density. Secreted and intracellular VWF antigen were measured by ELISA. The angiogenic capacity of ECFCs was investigated by the Matrigel tube formation assay. Differences in expression of genes involved in angiogenesis, aging, and endothelial to mesenchymal transition (EndoMT) were measured by quantitative PCR. Results Different ECFC lines show variable morphologies and cell density at maximum confluency and cell lines with a low maximum cell density show a mixed and more mesenchymal phenotype. We identified a significant positive correlation between maximum cell density and VWF production, both at protein and mRNA level. Also, significant correlations were observed between maximum cell density and several angiogenic, aging and EndoMT parameters. Conclusions We observed variations in morphology, maximum cell density, VWF production, and angiogenic potential between healthy control ECFCs. These variations seem to be attributable to differences in aging and EndoMT. Because variations correlate with cell density, we believe that ECFCs maintain a powerful tool to study vascular diseases. It is however important to compare cell lines with the same characteristics and perform experiments at maximum cell density.