title: In human endothelial cells rapamycin causes mTORC2 inhibition and impairs cell viability and function.
abstract: OBJECTIVE
  Drug-eluting stents are widely used to prevent restenosis but are associated with late endothelial damage. To understand the basis for this effect, we have studied the consequences of a prolonged incubation with rapamycin on the viability and functions of endothelial cells.
  
  
  RESULTS
  Human umbilical vein or aorta endothelial cells were exposed to rapamycin in the absence or in the presence of tumour necrosis factor alpha (TNFalpha). After a 24 h-incubation, rapamycin (100 nM) caused a significant cell loss associated with the increase of both apoptosis and necrosis, as quantified by propidium iodide staining, caspase 3 activity, and lactate dehydrogenase release. Rapamycin also impaired cell mobility, as assessed by a wound test, and promoted the formation of actin stress fibres, as determined with confocal microscopy. Moreover, the inhibitor prolonged TNFalpha-dependent E-selectin induction, inhibited endothelial nitric oxide synthase expression at both mRNA (quantitative real-time polymerase chain reaction) and protein level (enzyme-linked immunosorbent assay and western blot), and lowered bioactive nitric oxide output (RFL-6 reporter cell assay). Under the conditions adopted, rapamycin inhibited both mammalian target-of-rapamycin complexes (mTORC1 and mTORC2), as indicated by the reduced amount of raptor and rictor bound to mTOR in immunoprecipitates and by the marked hypophosphorylation of protein S6 kinase I (p70S6K) and Akt, determined by western blotting. The selective inhibition of mTORC1 by AICAR did not affect endothelial viability.
  
  
  CONCLUSIONS
  A prolonged treatment with rapamycin impairs endothelial function and hinders cell viability. Endothelial damage seems dependent on mTORC2 inhibition.
authors.affiliation: Department of Experimental Medicine, Unit of General and Clinical Pathology, University of Parma, Via Volturno 39, 43100 Parma, Italy.
authors.forename: Amelia
authors.initials: A
authors.lastname: Barilli
authors.forename: Rossana
authors.initials: R
authors.lastname: Visigalli
authors.forename: Roberto
authors.initials: R
authors.lastname: Sala
authors.forename: Gian C
authors.initials: GC
authors.lastname: Gazzola
authors.forename: Alessandro
authors.initials: A
authors.lastname: Parolari
authors.forename: Elena
authors.initials: E
authors.lastname: Tremoli
authors.forename: Sabrina
authors.initials: S
authors.lastname: Bonomini
authors.forename: Alexandra
authors.initials: A
authors.lastname: Simon
authors.forename: Ellen I
authors.initials: EI
authors.lastname: Closs
authors.forename: Valeria
authors.initials: V
authors.lastname: Dall'Asta
authors.forename: Ovidio
authors.initials: O
authors.lastname: Bussolati
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002317:Cardiovascular Agents / Q000633:toxicity*; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D019040:E-Selectin / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D047468:Immunoprecipitation; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D000076222:Mechanistic Target of Rapamycin Complex 1; D018613:Microscopy, Confocal; D046912:Multiprotein Complexes; D009336:Necrosis; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D000076223:Regulatory-Associated Protein of mTOR; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D020123:Sirolimus / Q000633:toxicity*; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases; D016559:Tacrolimus / Q000494:pharmacology; D013806:Theophylline / Q000031:analogs & derivatives; D013997:Time Factors; D014157:Transcription Factors / Q000037:antagonists & inhibitors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002317:Cardiovascular Agents; D002352:Carrier Proteins; D019040:E-Selectin; D046912:Multiprotein Complexes; D011506:Proteins; C492571:RICTOR protein, human; C463659:RPTOR protein, human; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D000076223:Regulatory-Associated Protein of mTOR; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D013806:Theophylline; D007770:L-Lactate Dehydrogenase; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D011494:Protein Kinases; C546842:MTOR protein, human; D000076222:Mechanistic Target of Rapamycin Complex 1; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020123:Sirolimus; D016559:Tacrolimus; C006683:bamifylline