Studying VWF secretion under flow using microfluidics and patient-derived endothelial cells

I. van Moort¹, Y. Sakurai², E. Hardy², P. Burgisser³, E. Vital², Y. Qiu², F. Leebeek³, W. Lam², R. Bierings⁴

¹Erasmus MC, University Medical Center Rotterdam, Rotterdam, Zuid-Holland, Netherlands, ²Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, USA & Department of Pediatrics, Division of Pediatric Hematology/Oncology, Aflac Cancer Center and Blood Disorders Center of Children’s Healthcare of Atlanta, Emory University School of Medicine, Atlanta, USA, Atlanta, Georgia, United States, ³Department of Hematology, Erasmus MC, University Medical Center Rotterdam, The Netherlands, Rotterdam, Zuid-Holland, Netherlands, ⁴Erasmus MC, University Medical Center Rotterdam, The Netherlands, Rotterdam, Zuid-Holland, Netherlands

Abstract Number: OC 53.1

Meeting: ISTH 2022 Congress

Theme: Platelet Disorders, von Willebrand Disease and Thrombotic Microangiopathies » von Willebrand Factor Biology

Background: Von Willebrand factor (VWF) is secreted from endothelial cells into the vessel lumen to form platelet adhesive strings at sites of vascular damage. Quantitative and qualitative defects in VWF lead to the bleeding disorder von Willebrand disease (VWD). Large inter-individual differences in bleeding phenotype exist in these patients that are currently not well understood but may be the result of variability in endothelial secretory responses. To study how endothelial secretion may affect the interplay with other blood components we need an ex vivo model to model the patient’s hemostatic response using patient endothelium under flow.

Aims: To model VWF secretion in patients with VWD using an ex vivo blood vessel consisting of a microfluidic channel endothelialized with patient-derived endothelial colony forming cells (ECFCs).

Methods: ECFCs were isolated from healthy volunteers and patients with VWD enrolled in the 2020-BOEK-MK study with informed consent. VWD patients were selected from the Willebrand in the Netherlands (WiN) study. PDMS microfluidic devices containing vascular channels (150µm x 50µm) were created using soft lithography. ECFCs were seeded in collagen-coated channels and cultured for 48h under flow (750s-1). To stimulate regulated secretion, ECFCs were stimulated under flow with 100µM histamine. VWF secretion was monitored using confocal microscopy.

Results: PDMS channels seeded with ECFCs formed 3D vessels consisting of confluent endothelial monolayers encapsulating a vessel lumen (figure 1). Immunostaining of VWF in healthy donor vessels revealed large numbers of Weibel-Palade bodies per cell. VWD 2A ECFCs (heterozygous p.C1190R) also formed 3D vessels, however significant amounts of VWF were retained in the endoplasmic reticulum. Histamine induced formation of numerous VWF strings covering the apical side of the endothelium facing the vessel lumen.

Conclusion(s): Microfluidic bleeding devices containing patient-derived ECFCs are promising models to study the cause of (variations in) bleeding abnormalities.

This study is funded by NWO-NWA.1160.18.038 and an ISTH-EHA Training Fellowship.

Figure 1

Vessel created from healthy ECFCs. A-VE-Cadherin; B- VWF; C- DAPI; D- merge with VE-Cadherin in Magenta, VWF in green, DAPI in blue.

To cite this abstract in AMA style:

van Moort I, Sakurai Y, Hardy E, Burgisser P, Vital E, Qiu Y, Leebeek F, Lam W, Bierings R. Studying VWF secretion under flow using microfluidics and patient-derived endothelial cells [abstract]. https://abstracts.isth.org/abstract/studying-vwf-secretion-under-flow-using-microfluidics-and-patient-derived-endothelial-cells/. Accessed October 1, 2023.

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