Everolimus-induced epithelial to mesenchymal transition (EMT) in bronchial/pulmonary cells: when the dosage does matter in transplantation

Background: Everolimus (EVE) is a mammalian target of rapamycin inhibitor (mTOR-I) widely used in transplantation that may determine some severe adverse events, including pulmonary fibrosis. The pathogenic mechanism of mTOR-I-associated pulmonary toxicity is still unclear, but epithelial to mesenchymal transition (EMT) of bronchial/pulmonary cells may play a role. Methods: Three cell lines—human type II pneumocyte-derived A549, normal bronchial epithelial, and bronchial epithelial homozygous for the delta F508 cystic fibrosis-causing mutation—were treated with EVE or tacrolimus at different concentrations. Real-time polymerase chain reaction and immunofluorescence were used to evaluate mRNA and protein levels of EMT markers (alpha-SMA, vimentin, fibronectin). Subsequently, in 13 EVE- and 13 tacrolimus-treated patients we compared the rate of lung fibrosis, estimated by an arbitrary pulmonary fibrosis index score (PFIS). Results: Biomolecular experiments demonstrated that high doses of EVE (100 nM) up-regulated EMT markers in all cell lines at both gene- and protein level. High concentrations of EVE were also able to reduce the mRNA levels of epithelial markers (E-cadherin and ZO-1) and to induce the phosphorylation of AKT. In the in vivo part of the study, PFIS was significantly higher in the EVE-group than the tacrolimus-group (p = 0.03) and correlated with trough levels (R2 = 0.35). Conclusions: Our data reveal, for the first time, a dose-dependent EVE-induced EMT in airway cells. They suggest that clinicians should employ, wherever possible, low dosages of mTOR-Is in transplant recipients, assessing periodically their pulmonary function.