Everolimus-induced Hyperpermeability of Endothelial Cells Causes Lung Injury

Background: The mammalian target of rapamycin (mTOR) inhibitors, everolimus (but not dactolisib), is frequently associated with lung injury in clinical therapies. However, the underlying mechanisms remain unclear. Endothelial cell barrier dysfunction plays a major role in the pathogenesis of the lung injury. This study hypothesizes that everolimus increases pulmonary endothelial permeability, which leads to lung injury. Methods： We tested the effects of everolimus and dactolisib on human pulmonary microvascular endothelial cell (HPMEC) permeability by measuring transendothelial electrical resistance (TEER) and the passage of horseradish peroxidase-albumin (Pa）. F-actin remodeling visualized using immunofluorescent imaging and its related key enzymes were measured by western blotting. Then, we established a mouse model using intraperitoneal injections of everolimus and dactolisib, and the total protein in bronchoalveolar lavage fluid (BALF) and the accumulation of Evans blue dye in lungs were used to examine the lung vascular permeability. The protective effects of myosin light chain (MLC) related pathway inhibitors on everolimus-induced hyperpermeability was observed. Results: Everolimus increased HPMEC permeability, which was confirmed by decreased TEER and an increased transendothelial BSA flux, which was associated with MLC phosphorylation and F-actin stress fiber formation. In contrast, dactolisib did not have effect on the permeability of HPMECs. In addition, inhibition, or knockdown of PKCα or MLC kinase attenuated everolimus-induced hyperpermeability and MLC phosphorylation in HPMECs. Furthermore, everolimus induced an increasing concentration of intracellular calcium Ca 2+ leakage in HPMECs and this was normalized with ryanodine pretreatment. Additionally, ryanodine decreased everolimus-induced phosphorylation of PKCα and MLC, and barrier disruption in HPMECs. Consistent with in vitro data, everolimus treatment caused a visible lung-vascular barrier dysfunction, including an increase in protein in BALF and lung capillary-endothelial permeability, which was significantly attenuated by pretreatment with an inhibitor of PKCα, MLCK, and ryanodine. Conclusions: This study shows that everolimus induced pulmonary endothelial hyper-permeability, at least partly, in an MLC phosphorylation-mediated EC contraction which is influenced in a Ca 2+ -dependent manner and can lead to lung injury.