In vitro exploration of drug-induced thrombotic microangiopathies: clues of diverse endothelial activation pathways respective to interferon-β1a, ciclosporin A, and gemcitabine exposure

Pro-inflammatory and pro-thrombotic stimuli can activate endothelial cells (ECs) and predispose them to thrombotic microangiopathies (TMAs). Drug-induced TMA (DITMA) may occur in clinical practice during treatment with interferon-β1a (IFN-β1a), ciclosporin A (CsA), and gemcitabine (GEM). DITMA may also trigger the complement system and induce membrane attack complex (MAC, C5b-9) deposition <i>in vivo</i>, although their role and the benefit of inhibition remain unclear. In an experimental <i>in vitro</i> model of microvascular ECs exposed to these three drugs, we searched for MAC deposits and drug-specific pro-inflammatory and pro-thrombotic traits to gain insights into the mechanisms potentially involved in DITMA. Human microvascular endothelial cells line-1 (HMEC-1) was treated with 10% normal human serum, CsA, GEM, and IFN-β1a. Cell viability for each drug was measured using the resazurin assay. Cell component expression of the following markers involved in endothelial pathogenic activation was measured via immunofluorescence and flow cytometry: C5b-9, interleukin (IL)-1α, IL-6, E-selectin, platelet EC adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), and von Willebrand factor (vWF). Levels of plasminogen activator inhibitor-1 (PAI-1) and urokinase plasminogen activator (uPA) were measured in the supernatants using the enzyme-linked immunosorbent assay (ELISA). Significantly increased C5b-9 deposits were found with each drug, and increased drug-specific activation marker expressions appeared in HMEC-1s when exposed to CsA (IL-1α, IL-6, ICAM-1, E-selectin, vWF, and uPA), GEM (IL-1α, IL-6, PECAM-1, ICAM-1, E-selectin, and vWF), and IFN-β1a (PECAM-1, ICAM-1, PAI-1, and uPA). Each drug induces MAC deposits on HMEC-1s and singular endothelial activation profiles, potentially leading to thrombogenesis observed in DITMA.