In human endothelial cells rapamycin causes mTORC2 inhibition and impairs cell viability and function

• Published in: Cardiovascular research Vol. 78; no. 3; pp. 563 - 571
• Main Authors: Barilli, Amelia, Visigalli, Rossana, Sala, Roberto, Gazzola, Gian C., Parolari, Alessandro, Tremoli, Elena, Bonomini, Sabrina, Simon, Alexandra, Closs, Ellen I., Dall’Asta, Valeria, Bussolati, Ovidio
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.06.2008
• Subjects: Adaptor Proteins, Signal Transducing; Antibacterial agents; Antibiotics. Antiinfectious agents. Antiparasitic agents; Apoptosis; Apoptosis - drug effects; Biological and medical sciences; Blotting, Western; Cardiology. Vascular system; Cardiovascular Agents - toxicity; Carrier Proteins - metabolism; Caspase 3 - metabolism; Cell Movement - drug effects; Cell Survival - drug effects; Dose-Response Relationship, Drug; E-Selectin - metabolism; Endothelial Cells - drug effects; Endothelial Cells - enzymology; Endothelial Cells - metabolism; Endothelial Cells - pathology; Enzyme-Linked Immunosorbent Assay; Humans; Immunoprecipitation; L-Lactate Dehydrogenase - metabolism; Mechanistic Target of Rapamycin Complex 1; Medical sciences; Microscopy, Confocal; mTOR; Multiprotein Complexes; Necrosis; Nitric oxide; Nitric Oxide - metabolism; Nitric Oxide Synthase Type III - metabolism; Pharmacology. Drug treatments; Polymerase Chain Reaction; Protein Kinases - metabolism; Proteins - metabolism; Rapamycin-Insensitive Companion of mTOR Protein; Regulatory-Associated Protein of mTOR; Restenosis; Ribosomal Protein S6 Kinases, 70-kDa - metabolism; Sirolimus - toxicity; Stress Fibers - drug effects; Stress Fibers - metabolism; Tacrolimus - pharmacology; Theophylline - analogs & derivatives; Time Factors; TNFalpha; TOR Serine-Threonine Kinases; Transcription Factors - antagonists & inhibitors; Tumor Necrosis Factor-alpha - metabolism; Up-Regulation; Restenosis; mTOR; Nitric oxide; TNFalpha; Apoptosis; Human; Endothelial cell; Cell function; TNF alpha; Sirolimus; Phlebology; Antibiotic; Tumor necrosis factor; Circulatory system; Inhibition; Antibacterial agent; Cardiology; Viability
• ISSN: 0008-6363; 1755-3245
• DOI: 10.1093/cvr/cvn024

Aim 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. Methods and results Human umbilical vein or aorta endothelial cells were exposed to rapamycin in the absence or in the presence of tumour necrosis factor α (TNFα). 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 TNFα-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. Conclusion A prolonged treatment with rapamycin impairs endothelial function and hinders cell viability. Endothelial damage seems dependent on mTORC2 inhibition.