Cyclosporin A toxicity on endothelial cells differentiated from induced pluripotent stem cells: Assembling an adverse outcome pathway.

Cyclosporin A (CSA) is a potent immunosuppressive agent in pharmacologic studies. However, there is evidence for side effects, specifically regarding vascular dysfunction. Its mode of action inducing endothelial cell toxicity is partially unclear, and a connection with an adverse outcome pathway (AOP) is not established yet. Therefore, we designed this study to get deeper insights into the mechanistic toxicology of CSA on angiogenesis. Stem cells, especially induced pluripotent stem cells (iPSCs) with the ability of differentiation to all organs of the body, are considered a promising in vitro model to reduce animal experimentation. In this study, we differentiated iPSCs to endothelial cells (ECs) as one cell type that in other studies would allow to generate multi-cell type organoids from single donors. Flow cytometry and immunostaining confirmed our scalable differentiation protocol. Then dose and time course experiments assessing CSA cytotoxicity on iPS derived endothelial cells were performed. Transcriptomic data suggested CSA dependent induction of reactive oxygen species (ROS), mitochondrial dysfunction, and impaired angiogenesis via ROS induction which was confirmed by in vitro experiments. In order to put these data into a potential adverse outcome pathway (AOP) context, we performed a literature review for CSA-mediated endothelial cell toxicity and combined our experimental data with the publicly available knowledge. Such an AOP will help to design in vitro test batteries and to model events observed in human toxicity studies, as well in predictive toxicology.