Optimization of disease- and patient-specific in vitro assays for endothelial dysfunction investigation by use of endothelial colony-forming cells

Background: The study of endothelial dysfunction (ED) is crucial to identify pathogenetic mechanism(s), define distinct clinicopathological entities and guide patient treatment in several pathological settings. Nevertheless, it is currently hindered by the limited availability of patient-specific primary cells. In this context, endothelial colony-forming cells (ECFCs) represent a valuable tool, as their characterization allows non-invasive functional assessment of the endothelial compartment. Aims: To introduce the use of ECFCs as a patient-specific primary cell source to analyze ED in a) thrombogenesis studies under flow conditions, and b) thrombin generation assay (TGA) on endothelial cells. Methods: Standard thrombogenesis assays and TGA were modified, using ECFCs as a substrate compared with commercially-available human umbilical venous endothelial cells (HUVECs) as a control. ECFCs were isolated from healthy donor peripheral blood using a protocol previously optimized in our lab. Endothelial function was assessed at passages 4-6, in basal conditions or after activation with TNFα, by analyzing: a) platelet deposition and fibrin formation under flow conditions, assessed in thrombogenesis assay by confocal microscopy; b) thrombin formation assessed by TGA on endothelial cells - a modification of Hamker’s method. Expression of the adhesion molecule CD106 (VCAM-1) and the pro-coagulant molecule Tissue Factor (TF) were also assessed, by flow cytometry. Results: The experimental setting of both assays was optimized on HUVECs and then tested on ECFCs, confirming on both cell types the ability of the assays to discriminate between unstimulated and activated cells. In fact, activated cells were characterized by a) increased platelet deposition and fibrin formation, and b) thrombin generation, compared with unstimulated cells. Activation of TNFα−treated endothelial cells was also confirmed by assessing the upregulation of CD106 and TF. Conclusion: Our results support the use of ECFCs for patient-specific in vitro investigation of ED, thus paving the way for their application in personalized modeling of vascular diseases.