Everolimus (EVL) is widely used in solid organ transplantation. It is known to have antiproliferative and immunosuppressive abilities via inhibition of the mTOR pathway. Preventive EVL administration may lower inflammation induced by cardiopulmonary bypass (CPB) and reduce systemic inflammatory response syndrome (SIRS). After oral loading with EVL 2.5 mg/kg/day (n = 11) or placebo (n = 11) for seven consecutive days, male Wistar rats (400-500 g) were connected to a miniaturised heart-lung-machine performing a deep hypothermic circulatory arrest protocol. White blood cells (WBC) were significantly reduced in EVL-pretreated animals before start of CPB with a preserved reduction by trend at all other time points. Ischemia/reperfusion led to decreased glucose levels. Application of EVL significantly increased glucose levels after reperfusion. In addition, potassium levels were significantly lower in EVL-treated animals at the end of reperfusion. Immunoblotting revealed increased S6 levels after CPB. EVL decreased phosphorylation of S6 in the heart and kidney, which indicates an inhibition of mTOR pathway. Moreover, EVL significantly modified phosphorylation of AKT, while decreasing IL2, IL6, RANTES, and TNFα (n = 6). Preventive application of EVL may modulate inflammation by inhibition of mammalian target of rapamycin (mTOR) pathway and reduction of proinflammatory cytokines. This may be beneficial to evade SIRS-related morbidities after CPB.

We investigated whether the inhibitory effect of the immunosuppressant everolimus (RAD001) on vascular smooth muscle cell (VSMC) proliferation is mediated by p27/kip1 gene promoter activity.

In this experimental study, cultured rat VSMCs were transiently transfected with a recombinant plasmid (pXp27) containing p27/kip1 gene promoter sequence and a chloramphenicol acetyltransferase (CAT) reporter gene. After stimulation with the mitogen platelet-derived growth factor (PDGF-BB, 10 ng/mL) in the presence or absence of RAD001 (10 nM), the promoter activity, mRNA expression, and protein expression of p27/kip1 were examined by CAT assay, RT-PCR, and immunoblotting, respectively. Cell cycle-related changes were detected by flow cytometry. DNA synthesis was determined using 3H-TdR incorporation.

Compared with the non-stimulation group, PDGF-BB stimulation induced a significant proliferative response in the VSMCs as indicated by decreased p27/kip1 gene promoter activity, decreased p27/kip1 mRNA and protein expression, increased S-phase and G2/M-phase cells, and increased DNA synthesis. RAD001 intervention increased p27/kip1 gene promoter activity 3.5-fold, promoted p27/kip1 mRNA and protein expression, increased G0-phase cells, reduced DNA synthesis, and, overall, inhibited PDGF-BB-stimulated cell proliferation.

RAD001 inhibits PDGF-BB-stimulated proliferation of cultured VSMCs by upregulating p27/kip1 gene promoter activity and increasing p27/kip1 mRNA and protein expression.

The present study aims to investigate the underlying mechanisms accounting for the activities of everolimus to inhibit the growth of vascular smooth muscle cells (VSMCs), which contributes to restenosis.

Primary VSMCs were cultured in media containing smooth muscle growth supplements and incubated with testing agents. Cell proliferation, cell cycle distribution, apoptosis and autophagy, and the key molecules involved, were examined.

Everolimus inhibited the proliferation of VSMCs by inhibiting the activation of ribosomal protein S6 kinase and phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1, and downregulating proliferating cellular nuclear antigen. Everolimus induced cell cycle arrest at the G1 phase by downregulating cyclin D1 and upregulating p27, and increased apoptosis by downregulating Bcl-2, upregulating Bad and activating capsase-3 and poly ADP ribose polymerase. Everolimus enhanced autophagy by increasing the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II, and upregulating Beclin 1. Specific autophagy inhibitors, 3-methyladenine and bafilomycin A1, significantly attenuated the inhibition of cell proliferation, the increased apoptosis and the altered expression of the above key proteins induced by everolimus.

Enhanced autophagy by everolimus contributes to its antirestenotic activity and its abilities to inhibit cell proliferation and to induce apoptosis of VSMCs.

Everolimus, a pharmaceutical component of drug-eluting stents, inhibits coronary vessel restenosis, but the antirestenotic mechanisms of action remain unclear. Here, we describe the effects of everolimus on key contributors to vessel restenosis, smooth muscle cell proliferation, and migration. In a dose-dependent fashion, everolimus reduced human coronary artery smooth muscle cell (HCASMC) proliferation without toxicity in a bimodal fashion, with accentuated potency occurring at 10 μM. Everolimus arrested the majority of HCASMCs in G1-phase, whereas it reduced the fraction of cells in S-phase at doses that inhibited DNA synthesis (bromodeoxyuridine incorporation). Consistent with this, Western blotting demonstrated that everolimus reduced activation and expression of G1-phase cell cycle progression factors, including p70S6K and cyclin D, respectively, decreased levels of proliferating cell nuclear antigen, and attenuated growth factor/serum-induced phosphorylation of the cell cycle phase transition intermediate, retinoblastoma protein. Everolimus did not, however, affect HCASMC migration. These observations suggest that everolimus acts as an antiproliferative, but not antimigratory, compound to account for at least some of the clinical efficacy exhibited by this drug as an antirestenotic agent. Moreover, everolimus-induced inhibition of the mammalian target of rapamycin complex 1 and regulation of cyclin-mediated cell cycle progression actions likely account for the antiproliferative effects of this compound on HCASMCs.

Statins, among the most commonly prescribed drugs worldwide, are cholesterol-lowering agents used to manage and prevent cardiovascular and coronary heart diseases. Recently, a multifaceted action in different physiological and pathological conditions has been also proposed for statins, beyond anti-inflammation and neuroprotection. Statins have been shown to act through cholesterol-dependent and -independent mechanisms and are able to affect several tissue functions and modulate specific signal transduction pathways that could account for statin pleiotropic effects. Typically, statins are prescribed in middle-aged or elderly patients in a therapeutic regimen covering a long life span during which metabolic processes, aging, and concomitant novel diseases, including cancer, could occur. In this context, safety, toxicity, interaction with other drugs, and the state of health have to be taken into account in subjects treated with statins. Some evidence has shown a dichotomous effect of statins with either cancer-inhibiting or -promoting effects. To date, clinical trials failed to demonstrate a reduced cancer occurrence in statin users and no sufficient data are available to define the long-term effects of statin use over a period of 10 years. Moreover, results from clinical trials performed to evaluate the therapeutic efficacy of statins in cancer did not suggest statin use as chemotherapeutic or adjuvant agents. Here, we reviewed the pharmacology of the statins, providing a comprehensive update of the current knowledge of their effects on tissues, biological processes, and pathological conditions, and we dissected the disappointing evidence on the possible future use of statin-based drugs in cancer therapy.

Mutational inactivation of the tumor suppressor tuberous sclerosis complex 2 (TSC2) constitutively activates mTORC1, increases cell proliferation, and induces the pathological manifestations observed in tuberous sclerosis (TS) and in pulmonary lymphangioleiomyomatosis (LAM). While the role of mTORC1 in TSC2-dependent growth has been extensively characterized, little is known about the role of mTORC2. Our data demonstrate that mTORC2 modulates TSC2-null cell proliferation and survival through RhoA GTPase and Bcl2 proteins. TSC2-null cell proliferation was inhibited not only by reexpression of TSC2 or small interfering RNA (siRNA)-induced downregulation of Rheb, mTOR, or raptor, but also by siRNA for rictor. Increased RhoA GTPase activity and P-Ser473 Akt were inhibited by siRNA for rictor. Importantly, constitutively active V14RhoA reversed growth inhibition induced by siRNA for rictor, siRNA TSC1, reexpression of TSC2, or simvastatin. While siRNA for RhoA had a modest effect on growth inhibition, downregulation of RhoA markedly increased TSC2-null cell apoptosis. Inhibition of RhoA activity downregulated antiapoptotic Bcl2 and upregulated proapoptotic Bim, Bok, and Puma. In vitro and in vivo, simvastatin alone or in combination with rapamycin inhibited cell growth and induced TSC2-null cell apoptosis, abrogated TSC2-null tumor growth, improved animal survival, and prevented tumor recurrence by inhibiting cell growth and promoting apoptosis. Our data demonstrate that mTORC2-dependent activation of RhoA is required for TSC2-null cell growth and survival and suggest that targeting both mTORC2 and mTORC1 by a combination of proapoptotic simvastatin and cytostatic rapamycin shows promise for combinational therapeutic intervention in diseases with TSC2 dysfunction.

Proliferation signal inhibitors (PSIs), everolimus (EVL), and sirolimus are a group of immunosuppressor agents indicated for the prevention of acute rejection in adult heart transplant recipients. Proliferation signal inhibitors have a mechanism of action with both immunosuppressive and antiproliferative effects, representing an especially interesting treatment option for the prevention and management of some specific conditions in heart transplant population, such as graft vasculopathy or malignancies. Proliferation signal inhibitors have been observed to work synergistically with calcineurin inhibitors (CNIs). Data from clinical trials and from the growing clinical experience show that when administered concomitantly with CNIs, PSIs allow significant dose reductions of the latter without loss of efficacy, a fact that has been associated with stabilization or significant improvement in renal function in patients with CNI-induced nephrotoxicity. The purpose of this article was to review the current knowledge of the role of PSIs in heart transplantation to provide recommendations for the proper use of EVL in cardiac transplant recipients, including indications, treatment regimens, monitoring, and management of the adverse events.

To investigate the effect of matrine on proliferation of vascular smooth muscle cells (VSMCs) and elucidate the underlying mechanisms.

Rat aortic VSMCs were cultured in medium supplemented with 10% fetal bovine serum and treated with various concentrations (0, 5, 10, 15, and 20 mg/L) of matrine for 72 h. VSMCs proliferation and cell cycle profiling were assessed using a methylene blue incorporation assay and flow cytometry, respectively. The underlying protein signaling mechanisms were determined using Western blot analysis of the expression levels of cell cycle regulatory genes, including p53, p21, p27, cyclin D1, cyclin E, cyclin-dependent kinase 2 and 4 (cdk2, cdk4), and phosphorylated Rb. The involvement of p21 and p27 pathways was further determined using small interfering RNA (siRNA) knockdown.

Matrine inhibited VSMC proliferation in a dose-dependent manner by promoting G(1) arrest. The G(1) arrest was accompanied by up-regulation of p53 and p21 protein levels, and down-regulation of cyclin D1/cdk4, cyclin E/cdk2 and phosphorylated Rb protein levels. Matrine did not affect p27 expression. Furthermore, the anti-proliferative effect of matrine was abolished by silencing of p21, but not by silencing of p27.

Our data indicate that matrine has an inhibitory effect on VSMC proliferation via up-regulation of the p53/p21 signaling pathway and modulation of other cell cycle regulatory genes.

Modulation of smooth muscle cells to a proliferating and migrating phenotype with downregulated alpha-actin expression is observed upon vascular lesion formation. The Id proteins (inhibitors of cell differentiation) play a role in the development of this phenotype. In contrast, synthetic peptides based on a conserved 11-residue Id sequence trigger the switch to a contractile phenotype that shows reduced cell growth and migration, increased expression of alpha-actin and decreased Id protein levels.