Standard immunosuppressive strategies after liver transplantation are based on steroids in combination with mycophenolic acid or calcineurin inhibitors (CNI); however their use might be associate with relevant side effects, most of all chronic renal failure, with an incidence of up to 20 % [
1]. The mTOR inhibitor and immunosuppressant Everolimus (EVE), in contrast with CNI, is associated with a low nephrotoxicity [
2]. EVE belongs to the rapamycin class of drugs that are allosteric inhibitors of mTORC1, an inhibitor of proliferative signal. The main mechanism of action of this drug is the inhibition of mTORC1 complex, a regulatory protein kinase involved in lymphocyte proliferation and other developmental processes [
3,
4]. EVE has gained wide interest also in other fields, for example, for the treatment of cancer, switching to less invasive phenotype of tumoral cells and inhibiting angiogenesis [
5,
6]. Then, due to this activity it has been proposed in de novo and maintenance liver transplant immunosuppressive protocols to prevent or treat hepatocarcinoma (HCC) recurrence, with survival benefits [
7,
8]. From the cellular point of view, interestingly, mTOR signaling is also involved in the mechanism of quiescent hepatic stellate cells (HSC) activation [
9]. For this reason, the potential role of mTOR-I in attenuating fibrogenic pathways has been already examined in experimental models, showing a reduced accumulation of extracellular matrix (ECM)-producing cells and ECM components [
10]. Then, in consideration of the role played by mTOR-I against proliferation and fibrogenesis, we can suppose that it could alleviate liver fibrosis also in the transplanted graft. Liver fibrosis, which is evident in 75 % of biopsies performed in long-term liver transplant (LTx) survivors, may be promoted by the recurrence of native disease (HCV), hepatotoxicity, de novo disease, non-alcoholic steatohepatitis, chronic rejection and vascular or biliary complications [
11]. However, despite evident clinical and experimental advantages of this drug category, mTOR-I may induce the development of several systemic side effects including hematological disorders (anemia, leukopenia and thrombocytopenia), dismetabolism (hyperlipidemia, post-transplant diabetes), lymphedema, stomatitis and fertility/gonadic toxicity [
12‐
14]. Fibrosis related pulmonary adverse effects (e.g., lymphocytic interstitial pneumonitis, bronchiolitis obliterans with organizing pneumonia and focal pulmonary fibrosis) have been also showed in the last years by several reports in oncological and renal transplant patients treated with mTOR-I [
15‐
18]. Our
in vitro experimental study has already evaluated novel cellular aspects of the potential pro-fibrotic activity of EVE in kidney, showing, for the first time, that epithelial to mesenchymal transition (EMT) in renal tubular cells may be activated by high doses of EVE [
19]. It is well known that EMT, a phenotypic conversion of epithelium to a fibroblastic or myofibroblastic phenotype, may have a pivotal role to induce fibrogenesis also in the liver microenviroment [
20]. In this context, rearranged ECM by migratory processes induced by EMT appears to be of particular importance also in the mechanism of HCC invasion. In fact, a key event in the development and progression of cancer is the potential of tumor cells to migrate and invade into surrounding tissues. As regards, involvement of PI3K/Akt/mTOR pathway in the interactions between HCC cells and non-parenchymal cells, such as mesenchymal stem cell (MSC), are critical issues for disease progression [
21]. Therefore, the aim of our study has been to analyze whether EVE at low-therapeutic [10 nM (corresponding to 3-8 ng/mL serum level)] and high (100 nM) doses might be able to induce in vitro EMT in human hepatoblastoma cells (HepG2) and to activate hepatic stellate cells (HSC). This work prompted us to assess the use of EVE in order to optimize its role in a tailored immunosuppressive regimen.