Variability of von Willebrand factor‐related parameters in endothelial colony forming cells

Abstract

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. AbstractBackgroundEndothelial 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.ObjectiveUnderstand the relation between phenotypic characteristics and VWF‐related parameters of healthy control ECFCs.MethodsECFC 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.ResultsDifferent 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.ConclusionsWe 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.

Ex vivo Improvement of a von Willebrand Disease Type 2A Phenotype Using an Allele-Specific Small-Interfering RNA

“…ECFCs are cultured endothelial cells that can be isolated from peripheral blood. 19,20 We show that siRNAs are able to selectively inhibit VWF alleles in healthy control ECFCs and that allele-specific siRNAs can improve the VWD phenotype of ECFCs that were isolated from a VWD type 2A patient with the VWF p.Cys1190Tyr mutation.…”

“…The medium was refreshed every other day until day 21. Passaging and usage of cells are performed as earlier described, 20 but with PromoCell culture medium. For the control ECFCs, a single clone was used for each experiment.…”

“…RNA isolation, cDNA synthesis, and quantitative polymerized chain reaction (qPCR) analysis with GAPDH as the endogenous reference gene were performed as described before. 20 qPCR was performed to quantify VWF, but also separate VWF alleles. Allele-specific qPCR primers were designed, containing the SNPs on the second last position of the forward primer.…”

“…ECFCs were fixated using ice-cold methanol and stained as described before. 20 Sheep anti-VWF (Abcam, ab11713), rabbit anti-protein disulphide isomerase (PDI; Stressgen Biotechnologies, San Diego, California, United States), and mouse anti-Golgi matrix protein of 130 kDa (GM130, BD Biosciences) were used as primary antibodies.…”

“…29 However, also clear variations between various ECFC cell lines emerge. 20 Most importantly, clear variations in VWF expression exist between ECFC lines and it was therefore suggested to be cautious when comparing different ECFC lines. 20 When allele-specific siRNAs are tested in (patient-derived) ECFCs, the effects are determined in ECFCs transfected with either an allele-specific siRNA or a negative control siRNA.…”