Optimizing Assays to Characterize Endothelial Dysfunction in Patients with Antiphospholipid Syndrome

Background Antiphospholipid syndrome (APS) is a prothrombotic autoimmune disease that occurs as primary condition (PAPS) or associated with other disease(s). APS diagnosis requires at least one clinical criterion (thrombosis and/or pregnancy morbidity) in the presence of antiphospholipid antibodies (aPLs). aPLs can be also found in healthy individuals (aPL carriers). The endothelial dysfunction (ED) that results from the interaction between aPL and endothelial cells (ECs) is a contributor to the development of APS clinical manifestations. However, it is not yet known whether constitutive ED may further predispose the host to the development of the disease. In this context, patient-specific endothelial colony-forming cells (ECFCs) are a valuable tool to investigate ED. Aim To develop assays to evaluate ED in patient-specific ECFCs. Methods A fluorescence microscopy adhesion assay and a seven-color flow cytometry panel aimed to assess ED were developed on unstimulated and TNFα-treated primary ECs (HUVECs). The developed FACS panel was then applied to assess ED on ECFCs isolated from PAPS patients, aPL carriers, and healthy donors (HDs). Results The adhesion assay discriminated between activated and resting ECs as demonstrated by higher leukocyte adhesion on TNFα-treated than unstimulated HUVECs. The FACS panel allowed to assess EC activation, as demonstrated by an increased expression of the adhesion molecules VCAM-1, ICAM-1 and the procoagulant molecule TF, associated with a decreased expression of the anticoagulant molecules TM and EPCR on TNFα-treated than unstimulated cells. Based on the expression levels of these markers, PAPS ECFCs resulted more activated than HD ECFCs. Conclusions The developed assays effectively distinguish between activated and resting ECs. Their application – as suggested by the more activated phenotype of PAPS ECFCs observed in flow cytometry – will be a valuable strategy to investigate in depth ED in APS patients.