A multilevel approach to dissect constitutive endothelial dysfunction in patient derived endothelial colony-forming cells

Endothelial dysfunction (ED) is a key contributor to thromboinflammatory processes, yet its mechanistic characterization in patients remains challenging. Endothelial colony-forming cells (ECFC), isolated from peripheral blood, provide a unique platform to investigate constitutive alterations in the patient-derived endothelium.​ We established a comprehensive multilevel approach to interrogate ED. Phenotypic characterization of ECFC was performed by multiparametric flow cytometry, focusing on the balance between procoagulant and anticoagulant molecules, as well as on adhesion receptors mediating leukocyte- and platelet–endothelium interactions. In parallel, soluble endothelial markers—encompassing adhesion molecules, selectins, and cell–cell interaction mediators—were quantified both in ECFC supernatants and in matched patient plasma to provide accessible correlates of endothelial activation. To assess the role of ED in the thrombotic process and specifically investigate the prothrombotic activity of ECFCs, we developed a modified thrombin generation assay (TGA) to measure thrombin formation, a key step in fibrin generation. Additionally, we adapted a microfluidic thrombogenesis assay in which healthy donor whole blood was perfused over ECFC monolayers to evaluate platelet adhesion, representing the initial phase of hemostasis, and fibrin formation, the final phase, under controlled shear conditions.​ This multilevel strategy enables a systematic assessment of constitutive ED, linking molecular signatures with functional consequences across the coagulation cascade. Importantly, we successfully applied this approach in different clinical contexts. In unprovoked venous thromboembolism (VTE), it revealed the presence of constitutive ED, which was not observed in provoked or weakly provoked VTE patients. In antiphospholipid syndrome (APS), it highlighted a more pronounced ED in patients with thrombotic manifestations compared to those with obstetric complications. Together, these applications underscore the strength of our methodology as a versatile framework for dissecting disease-related endothelial alterations and for uncovering mechanistic pathways relevant to vascular pathology.​