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

Abstract

Cyclosporin A (CSA) is a potent immunosuppressive agent in pharmacologic studies. However, there is evidence for side effects, specifically in regard to 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 cells or 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 CDA dependent induction of reactive oxygen species (ROS) and mitochondrial dysfunction, which was confirmed by in vitro experiments. Additionally, CSA impaired angiogenesis via ROS induction. Finally, we combined this information into an AOP, was developed based on here observed and literature based evidence for CSA-mediated endothelial cell toxicity. This AOP will help to design in vitro test batteries, model events observed in human toxicity studies, as well for predictive toxicology.