Sandimmune induced endothelial dysfunction: Differential actions of cyclosporin and cremaphor

Abstract Sandimmune (cyclosporin emulsion) is widely used in immunosuppressive therapy following organ transplant. However, Sandimmune therapy is associated with a high incidence of end-organ damage. In this study, we hypothesized that Sandimmune therapy causes endothelial cell dysfunction leading to disruption of cell--cell integrity in vitro . Further, we investigated the cellular mechanisms through which this damage may occur. Human aortic endothelial cells (10 4 cells) were seeded onto Matrigel and treated with 10 μg/ml cyclosporin/cremaphor (Sandimmune), 10 μg/ml cyclosporin/DMSO, 10 μl/ml cremaphor, or 10 μl/ml DMSO ( n = 9, for all groups). The cells were then incubated for 24 h in 95% O 2 /5% CO 2 at 37° and the degree of tube formation determined by photomicroscopy followed by computerized image analysis. In another set of experiments, we allowed the endothelial tubes to form over a period of 24 h followed by treatment with 10 μg/ml cyclosporin/cremaphor (Sandimmune), 10 μg/ml cyclosporin/DMSO, 10 μl/ml cremaphor, or 10 μl/ml DMSO, then incubated in 95% O 2 /5% CO 2 at 37° for 24 h ( n = 9 for all groups). The degree of tube disruption was determined by photomicroscopy followed by computerized image analysis. The results of these experiments demonstrate that cyclosporin whether solubilized in cremaphor or DMSO both inhibits the formation of endothelial tubes and causes disruption of previously formed endothelial tubes, while DMSO alone has no effect. Further, exposure of endothelial cells to cremaphor alone inhibits the formation of endothelial tubes and disrupts previously formed tubes. We next attempted to determine the mechanism(s) of endothelial dysfunction mediated by both cyclosporin and cremaphor. Microarray analysis of endothelial cells incubated with cremaphor demonstrated a significant increase (8.8 fold, n = 9) in the expression of the transcription factor HEY-1, which has been previously shown to be important in the regulation of angiogenesis. Cyclosporin/DMSO treatment of endothelial cells exhibited no change in the expression of HEY-1; however, when cells were incubated with cyclosporin/cremaphor, the expression of HEY-1 rose to 16-fold above control ( n = 9). Given that cyclosporin and cremaphor had a differential effect on HEY-1 expression, we decided to examine what other mechanism(s) may be responsible for the effect of cyclosporin on endothelial tube integrity. We have previously demonstrated the cyclosporin increases the expression of endothelin-1. This observation, coupled with studies in other labs indicating that cyclosporin increases oxidative stress in endothelial cells, led us to examine the role of NADPH oxidase, Caspase-8, and P38 MAP kinase in the effect of cyclosporin on endothelial cells. Preliminary studies indicate that these proteins may play a role in the disruptive effect of cyclosporin/DMSO on endothelial cells. Taken together, these results demonstrate that cyclosporin and cremaphor cause endothelial dysfunction through different mechanisms, although cyclosporin exhibited a permissive effect on the cremaphor-induced expression of HEY-1. Understanding the mechanism(s) of endothelial dysfunction mediated by the separate components of Sandimmune may lead to the development of safer immunosuppressive therapies.