Transcriptional and functional profiling identifies inflammation and endothelial-to-mesenchymal transition as potential drivers for phenotypic heterogeneity within a cohort of endothelial colony forming cells

Sebastiaan N J Laan; Suzan de Boer; Richard J Dirven; Iris van Moort; Thomas B Kuipers; Hailiang Mei; Ruben Bierings; Jeroen Eikenboom; SYMPHONY consortium

doi:10.1016/j.jtha.2024.03.018

Transcriptional and functional profiling identifies inflammation and endothelial-to-mesenchymal transition as potential drivers for phenotypic heterogeneity within a cohort of endothelial colony forming cells

J Thromb Haemost. 2024 Apr 2:S1538-7836(24)00176-4. doi: 10.1016/j.jtha.2024.03.018. Online ahead of print.

Authors

Sebastiaan N J Laan¹, Suzan de Boer², Richard J Dirven², Iris van Moort³, Thomas B Kuipers⁴, Hailiang Mei⁴, Ruben Bierings³, Jeroen Eikenboom⁵; SYMPHONY consortium

Collaborators

SYMPHONY consortium:
Martijn Brands, Sjoerd Koopman, Laura Bukkems, Michael Cloesmeijer, Alexander Janssen, Karin Fijnvandraat, Samantha Gouw, Ron Mathôt, Lotte Haverman, Emile van den Akker, Maartje van den Biggelaar, Masja de Haas, Sander Meijer, Jan Voorberg, Jessica Del Castillo Alferez, Huan Zhang, Johan Boender, Stephan Meijer, Karina Meijer, Sean de Jong, Geertje Goedhart, Anske van der Bom, Mettine Bos, Jeroen Eikenboom, Felix van der Meer, Sebastiaan Laan, Saskia Schols, Ruben Bierings, Lex Burdorf, Marjon Cnossen, Jan Hazelzet, Elise Huisman, Marieke Kruip, Frank Leebeek, Nikki van Leeuwen, Hester Lingsma, Moniek de Maat, Iris van Moort, Suzanne Polinder, Simone Reitsma, Eliza Roest, Ryanne Arisz, Lorenzo Romano, Wala Al Arashi, Shannon van Hoorn, Tine Goedhart, Caroline Mussert, Diaz Prameyllawati, Carin Uyl, Nathalie Jansen, Kathelijn Fischer, Hans Kristian Ploos van Amstel, Rolf Urbanus, Minka Zivkovic, Annelien Bredenoord, Rieke van der Graaf, Lieke Baas, Roger Schutgens, Mariëtte Driessens

Affiliations

¹ Department of Internal Medicine, Division of Thrombosis and Hemostasis, Leiden University Medical Centre, Leiden, the Netherlands; Department of Hematology, Erasmus University Medical Centre, Rotterdam, the Netherlands. Electronic address: https://twitter.com/laan_bas.
² Department of Internal Medicine, Division of Thrombosis and Hemostasis, Leiden University Medical Centre, Leiden, the Netherlands.
³ Department of Hematology, Erasmus University Medical Centre, Rotterdam, the Netherlands.
⁴ Sequencing Analysis Support Core, Department of Biomedical Data Sciences, Leiden University Medical Centre, Leiden, the Netherlands.
⁵ Department of Internal Medicine, Division of Thrombosis and Hemostasis, Leiden University Medical Centre, Leiden, the Netherlands. Electronic address: h.c.j.eikenboom@lumc.nl.

PMID: 38574861
DOI: 10.1016/j.jtha.2024.03.018

Abstract

Background: Endothelial colony-forming cells (ECFCs) derived from patients can be used to investigate pathogenic mechanisms of vascular diseases like von Willebrand disease. Considerable phenotypic heterogeneity has been observed between ECFC clones derived from healthy donors. This heterogeneity needs to be well understood in order to use ECFCs as endothelial models for disease.

Objectives: Therefore, we aimed to determine phenotypic and gene expression differences between control ECFCs.

Methods: A total of 34 ECFC clones derived from 16 healthy controls were analyzed. The transcriptome of a selection of ECFC clones (n = 15) was analyzed by bulk RNA sequencing and gene set enrichment analysis. Gene expression was measured in all ECFC clones by quantitative polymerase chain reaction. Phenotypic profiling was performed and migration speed of the ECFCs was measured using confocal microscopy, followed by automated quantification of cell morphometrics and migration speed.

Results: Through hierarchical clustering of RNA expression profiles, we could distinguish 2 major clusters within the ECFC cohort. Major differences were associated with proliferation and migration in cluster 1 and inflammation and endothelial-to-mesenchymal transition in cluster 2. Phenotypic profiling showed significantly more and smaller ECFCs in cluster 1, which contained more and longer Weibel-Palade bodies. Migration speed in cluster 1 was also significantly higher.

Conclusion: We observed a range of different RNA expression patterns between ECFC clones, mostly associated with inflammation and clear differences in Weibel-Palade body count and structure. We developed a quantitative polymerase chain reaction panel that can be used for the characterization of ECFC clones, which is essential for the correct analysis of pathogenic mechanisms in vascular disorders.

Keywords: RNA-seq; Weibel–Palade bodies; cell migration; endothelial cells; hemostasis; von Willebrand factor.