The Journal of Heart and Lung Transplantation
International Society for Heart and Lung Transplantation.

mTOR Inhibitors Ameliorate Ischemia-Reperfusion Injury through Endothelin-A Modulation and Reduction of Free Radical Production


      mTOR inhibitors are immunosuppressive agents used in maintenance immunosuppression. Everolimus and Sirolimus significantly influence the transmigration of immune cells after ischemia/reperfusion injury (I/R), as we showed before. This fact could explain a reduction of graft infiltration after early immunosuppressive treatment with mTOR inhibitors. The aim of our study was to investigate the underlying mechanisms by which Everolimus and Sirolimus are able to reduce I/R.


      An I/R model with human microvascular EC and human circulating immune cells (PBMC) was designed to evaluate reactive oxygen species (ROS) expression of EC and Endothelin-1 secretion of EC. For the analysis, ECs were either in naïve condition or activated with IFN-γ /TNF-α for 24h. After cell activation EC were placed under hypoxic conditions (<2% O2) for 2h and were further treated before re-oxygenation with Everolimus (10ng/ml Certican® Novartis, Basel, Switzerland) or Sirolimus (10 ng/ml Rapamune, Pfizer, NY, USA) for 2 and 24h. Untreated cells served as control and hypoxic cells served as positive control.


      The exposure of EC to I/R caused a significant increase of ROS levels, especially in activated ECs. Treatment of naïve EC with Everolimus and Sirolimus for 2 h could prevent the upregulation of ROS production compared to the prolonged 24 h EC treatment (naïve-EC Everolimus: 2h 1.75±0.07 FI vs. 24h 2.11±0.14 FI; naïve-EC Sirolimus: 2h 1.47±0.06 FI vs. 24h 1.88±0.09 FI). Prolonged Everolimus and Sirolimus treatment significantly reduced endothelin-1 secretion of activated EC (act-EC/act-PBMC Everolimus: 2h 26.0±1.0 pg/ml vs. 24h 16.2±0.6 pg/ml; act-EC/act-PBMC Sirolimus: 2h 25.9±0.8 pg/ml vs. 24h 16.0±1.2 pg/ml; act-EC/naive-PBMC Everolimus: 2h 22.2±0.4 pg/ml vs. 24h 11.7±0.8 pg/ml; act-EC/naive-PBMC Sirolimus: 2h 24.5±0.6 pg/ml vs. 24h 13.7±0.4 pg/ml).


      mTOR inhibitors are able to reduce ROS after I/R. It seems that the protective effect diminishes within short time and the application should already be started previous to severe inflammation. Everolimus and Sirolimus are also able to reduce Endothelin-1 production. In case of inflammation long time treatment with both immunosuppressive agents can positively affect Endothelin-1 levels, preventing endothelial inflammation.